Streptococcus equisimilis hyaluronan synthase gene and expression thereof in Bacillus subtilis

ABSTRACT

The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active  Streptococcus equisimilis  hyaluronate synthase (seHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/899,040, filed Jul. 23, 1997, entitled “HYALURONATE SYNTHASE GENE ANDUSES THEREOF”, and relates to U.S. Provisional Application U.S. Ser. No.60/064,435, filed Oct. 31, 1997, entitled “GROUP C HYALURONAN SYNTHASEGENE AND USES THEREOF”.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The government owns certain rights in the present inventionpursuant to grant number GM35978 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to a nucleic acid segment having acoding region segment encoding enzymatically active Streptococcusequisimilis hyaluronate synthase (seHAS), and to the use of this nucleicacid segment in the preparation of recombinant cells which producehyaluronate synthase and its hyaluronic acid product. Hyaluronate isalso known as hyaluronic acid or hyaluronan.

[0005] 2. Brief Description of the Related Art

[0006] The incidence of streptococcal infections is a major health andeconomic problem worldwide, particularly in developing countries. Onereason for this is due to the ability of Streptococcal bacteria to growundetected by the body's phagocytic cells, i.e., macrophages andpolymorphonuclear cells (PMNs). These cells are responsible forrecognizing and engulfing foreign microorganisms. One effective way thebacteria evade surveillance is by coating themselves with polysaccharidecapsules, such as a hyaluronic acid (HA) capsule. The structure of HA isidentical in both prokaryotes and eukaryotes. Since HA is generallynonimmunogenic, the encapsulated bacteria do not elicit an immuneresponse and are, therefore, not targeted for destruction. Moreover, thecapsule exerts an antiphagocytic effect on PMNs in vitro and preventsattachment of Streptococcus to macrophages. Precisely because of this,in Group A and Group C Streptococci, the HA capsules are major virulencefactors in natural and experimental infections. Group A Streptococcusare responsible for numerous human diseases including pharyngitis,impetigo, deep tissue infections, rheumatic fever and a toxic shock-likesyndrome. The Group C Streptococcus equisimilis is responsible forosteomyelitis, pharyngitis, brain abscesses, and pneumonia.

[0007] Structurally, HA is a high molecular weight linear polysaccharideof repeating disaccharide units consisting of N-acetylglucosamine(GlcNAc) and glucuronic acid (GlcA). The number of repeatingdisaccharides in an HA molecule can exceed 30,000, a M_(r)>10⁷. HA isthe only glycosaminogylcan synthesized by both mammalian and bacterialcells particularly Groups A and C Streptococci and Type A Pasturellamultocida. These strains make HA which is secreted into the medium aswell as HA capsules. The mechanism by which these bacteria synthesize HAis of broad interest medicinally since the production of the HA capsuleis a very efficient and clever way that Streptococci use to evadesurveillance by the immune system.

[0008] HA is synthesized by mammalian and bacterial cells by the enzymehyaluronate synthase which has been localized to the plasma membrane. Itis believed that the synthesis of HA in these organisms is a multi-stepprocess. Initiation involves binding of an initial precursor, UDP-GlcNAcor UDP-GlcA. This is followed by elongation which involves alternateaddition of the two sugars to the growing oligosaccharide chain. Thegrowing polymer is extruded across the plasma membrane region of thecell and into the extracellular space. Although the HA biosyntheticsystem was one of the first membrane heteropolysaccharide syntheticpathways studied, the mechanism of HA synthesis is still not wellunderstood. This may be because in vitro systems developed to date areinadequate in that de novo biosynthesis of HA has not been accomplished.

[0009] The direction of HA polymer growth is still a matter ofdisagreement among those of ordinary skill in the art. Addition of themonosaccharides could be to the reducing or nonreducing end of thegrowing HA chain. Furthermore, questions remain concerning (i) whethernascent chains are linked covalently to a protein, to UDP or to a lipidintermediate, (ii) whether chains are initiated using a primer, and(iii) the mechanism by which the mature polymer is extruded through theplasma membrane of the Streptococcus. Understanding the mechanism of HAbiosynthesis may allow development of alternative strategies to controlStreptococcal and Pasturella infections by interfering in the process.

[0010] HA has been identified in virtually every tissue in vertebratesand has achieved widespread use in various clinical applications, mostnotably and appropriately as an intra-articular matrix supplement and ineye surgery. The scientific literature has also shown a transition fromthe original perception that HA is primarily a passive structuralcomponent in the matrix of a few connective tissues and in the capsuleof certain strains of bacteria to a recognition that this ubiquitousmacromolecule is dynamically involved in many biological processes: frommodulating cell migration and differentiation during embryogenesis toregulation of extracellular matrix organization and metabolism toimportant roles in the complex processes of metastasis, wound healing,and inflammation. Further, it is becoming clear that HA is highlymetabolically active and that cells focus much attention on theprocesses of its synthesis and catabolism. For example, the half-life ofHA in tissues ranges from 1 to 3 weeks in cartilage to <1 day inepidermis.

[0011] It is now clear that a single protein utilizes both sugarsubstrates to synthesize HA. The abbreviation HAS, for the HA synthase,has gained widespread support for designating this class of enzymes.Markovitz et al. successfully characterized the HAS activity fromStreptococcus pyogenes and discovered the enzymes's membranelocalization and its requirements for sugar nucleotide precursors andMg²⁺. Prehm found that elongating HA, made by B6 cells, was digested byhyaluronidase added to the medium and proposed that HAS resides at theplasma membrane. Philipson and Schwartz also showed that HAS activitycofractionated with plasma membrane markers in mouse oligodendrogliomacells.

[0012] HAS assembles high M_(r) HA that is simultaneously extrudedthrough the membrane into the extracellular space (or to make the cellcapsule in the case of bacteria) as glycosaminoglycan synthesisproceeds. This mode of biosynthesis is unique among macromolecules sincenucleic acids, proteins, and lipids are synthesized in the nucleus,endoplasmic reticulum/Golgi, cytoplasm, or mitochondria. The extrusionof the growing chain into the extracellular space also allows forunconstrained polymer growth, thereby achieving the exceptionally largesize of HA, whereas confinement of synthesis within a Golgi orpost-Golgi compartment could limit the overall amount or length of thepolymers formed. High concentrations of HA within a confined lumen couldalso create a high viscosity environment that might be deleterious forother organelle functions.

[0013] Several studies attempted to solubilize, identify, and purify HASfrom strains of Streptococci that make a capsular coat of HA as well asfrom eukaryotic cells. Although the streptococcal and murineoligodendroglioma enzymes were successfully detergent-solubilized andstudied, efforts to purify an active HAS for further study or molecularcloning remained unsuccessful for decades. Prehm and Mausolf usedperiodate-oxidized UDP-GlcA or UDP-GlcNAc to affinity label a protein of˜52 kDa in streptococcal membranes that co-purified with HAS. This ledto a report claiming that the Group C streptococcal HAS had been cloned,which was unfortunately erroneous. This study failed to demonstrateexpression of an active synthase and may have actually cloned a peptidetransporter. Triscott and van de Rijn used digitonin to solubilize HASfrom streptococcal membranes in an active form. Van de Rijn and Drakeselectively radiolabeled three streptococcal membrane proteins of 42,33, and 27 kDa with 5-azido-UDP-GlcA and suggested that the 33-kDaprotein was HAS. As shown later, however, HAS actually turned out to bethe 42-kDa protein.

[0014] Despite these efforts, progress in understanding the regulationand mechanisms of HA synthesis was essentially stalled, since there wereno molecular probes for HAS mRNA or HAS protein. A major breakthroughoccurred in 1993 when DeAngelis et al. reported the molecular cloningand characterization of the Group A streptococcal gene encoding theprotein HasA. This gene was known to be in part of an operon requiredfor bacterial HA synthesis, although the function of this protein, whichis now designated as spHAS (the S. pyogenes HAS), was unknown. spHAS wassubsequently proven to be responsible for HA elongation and was thefirst glycosaminoglycan synthase identified and cloned and thensuccessfully expressed. The S. pyogenes HA synthesis operon encodes twoother proteins. HasB is a UDP-glucose dehydrogenase, which is requiredto convert UDP-glucose to UDP-GlcA, one of the substrates for HAsynthesis. HasC is a UDP-glucose pyrophosphorylase, which is required toconvert glucose 1-phosphate and UTP to UDP-glucose. Co-transfection ofboth hasA and hasB genes into either acapsular Streptococcus strains orEnteroccus faecalis conferred them with the ability to synthesize HA andform a capsule. This provided the first strong evidence that HasA is anHA synthase.

[0015] The elusive HA synthase gene was finally cloned by a transposonmutagenesis approach, in which an acapsular mutant Group A strain wascreated containing a transposon interruption of the HA synthesis operon.Known sequences of the transposon allowed the region of the junctionwith streptococcal DNA to be identified and then cloned from wild-typecells. The encoded spHAS was 5-10% identical to a family of yeast chitinsynthases and 30% identical to the Xenopus laevis protein DG42(developmentally expressed during gastrulation), whose function wasunknown at the time. DeAngelis and Weigel expressed the activerecombinant spHAS in Escherichia coli and showed that this singlepurified gene product synthesizes high M_(r) HA when incubated in vitrowith UDP-GlcA and UDP-GlcNAc, thereby showing that bothglycosyltransferase activities required for HA synthesis are catalyzedby the same protein, as first proposed in 1959. This set the stage forthe almost simultaneous identification of eukaryotic HAS cDNAs in 1996by four laboratories revealing that HAS is a multigene family encodingdistinct isozymes. Two genes (HAS1 and HAS2) were quickly discovered inmammals (29-34), and a third gene HAS3 was later discovered. A secondstreptococcal seHAS or Streptococcus equisimilis hyaluronate synthase,has now been found and is the invention being claimed and disclosedherein.

[0016] As indicated, we have also identified the authentic HAS gene fromGroup C Streptococcus equisimilis (seHAS); the seHAS protein has a highlevel of identity (approximately 70 percent) to the spHAS enzyme. Thisidentity, however, is interesting because the seHAS gene does notcross-hybridize to the spHAS gene.

[0017] Membranes prepared from E. coli expressing recombinant seHASsynthesize HA when both substrates are provided. The results confirmthat the earlier report of Lansing et al. claiming to have cloned theGroup C HAS was wrong. Unfortunately, several studies have employedantibody to this uncharacterized 52-kDa streptococcal protein toinvestigate what was believed to be eukaryotic HAS.

[0018] Itano and Kimata used expression cloning in a mutant mousemammary carcinoma cell line, unable to synthesize HA, to clone the firstputative mammalian HAS cDNA (mmHAS1). Subclones defective in HAsynthesis fell into three separate classes that were complementary forHA synthesis in somatic cell fusion experiments, suggesting that atleast three proteins are required. Two of these classes maintained someHA synthetic activity, whereas one showed none. The latter cell line wasused in transient transfection experiments with cDNA prepared from theparental cells to identify a single protein that restored HA syntheticactivity. Sequence analyses revealed a deduced primary structure for aprotein of ˜65 kDa with a predicted membrane topology similar to that ofspHAS. mmHAS1 is 30% identical to spHAS and 55% identical to DG42. Thesame month this report appeared, three other groups submitted papersdescribing cDNAs encoding what was initially thought to be the samemouse and human enzyme. However, through an extraordinary circumstance,each of the four laboratories had discovered a separate HAS isozyme inboth species.

[0019] Using a similar functional cloning approach to that of Itano andKimata, Shyjan et al. identified the human homolog of HAS 1. Amesenteric lymph node cDNA library was used to transfect murine mucosalT lymphocytes that were then screened for their ability to adhere in arosette assay. Adhesion of one transfectant was inhibited by antisera toCD44, a known cell surface HA-binding protein, and was abrogateddirectly by pretreatment with hyaluronidase. Thus, rosetting by thistransfectant required synthesis of HA. Cloning and sequencing of theresponsible cDNA identified hsHAS1. Itano and Kimata also reported ahuman HAS1 cDNA isolated from a fetal brain library. The hsHAS1 cDNAsreported by the two groups, however, differ in length; they encode a 578or a 543 amino acid protein. HAS activity has only been demonstrated forthe longer form.

[0020] Based on the molecular identification of spHAS as an authentic HAsynthase and regions of near identity among DG42, spHAS, and NodC (aβ-GlcNAc transferase nodulation factor in Rhizobium), Spicer et al. useda degenerate RT-PCR approach to clone a mouse embryo cDNA encoding asecond distinct enzyme, which is designated mmHAS2. Transfection ofmmHAS2 cDNA into COS cells directed de novo production of an HA cellcoat detected by a particle exclusion assay, thereby providing strongevidence that the HAS2 protein can synthesize HA. Using a similarapproach, Watanabe and Yamaguchi screened a human fetal brain cDNAlibrary to identify hsHAS2. Fulop et al. independently used a similarstrategy to identify mmHAS2 in RNA isolated from ovarian cumulus cellsactively synthesizing HA, a critical process for normal cumulus oophorusexpansion in the pre-ovulatory follicle. Cumulus cell-oocyte complexeswere isolated from mice immediately after initiating an ovulatory cycle,before HA synthesis begins, and at later times when HA synthesis is justbeginning (3 h) or already apparent (4 h). RT-PCR showed that HAS2 mRNAwas absent initially but expressed at high levels 3-4 h later suggestingthat transcription of HAS2 regulates HA synthesis in this process. BothhsHAS2 are 552 amino acids in length and are 98% identical. mmHAS1 is583 amino acids long an 95% identical to hsHAS1, which is 578 aminoacids long.

[0021] Most recently Spicer et al. used a PCR approach to identify athird HAS gene in mammals. The mmHAS3 protein is 554 amino acids longand 71, 56, and 28% identical, respectively, to mmHAS1, mmHAS2, DG42,and spHAS. Spicer et al. have also localized the three human and mousegenes to three different chromosomes (HAS1 to hsChr 19/mmChr 17; HAS2 tohsChr 8/mmChr 15; HAS3 to hsChr 16/mmChr 8). Localization of the threeHAS genes on different chromosomes and the appearance of HA throughoutthe vertebrate class suggest that this gene family is ancient and thatisozymes appeared by duplication early in the evolution of vertebrates.The high identity (˜30%) between the bacterial and eukaryotic HASs alsosuggests that the two had a common ancestral gene. Perhaps primitivebacteria usurped the HAS gene from an early vertebrate ancestor beforethe eukaryotic gene products became larger and more complex.Alternatively, the bacteria could have obtained a larger vertebrate HASgene and deleted regulatory sequences nonessential for enzyme activity.

[0022] The discovery of X. laevis DG42 by Dawid and co-workers played asignificant role in these recent developments, even though this proteinwas not known to be an HA synthase. Nonetheless, that DG42 and spHASwere 30% identical was critical for designing oligonucleotides thatallowed identification of mammalian HAS2. Ironically, definitiveevidence that DG42 is a bona fide HA synthase was reported only afterthe discoveries of the Mammalian isozymes, when DeAngelis and Achyuthanexpressed the recombinant protein in yeast (an organism that cannotsynthesize HA) and showed that it synthesizes HA when isolated membranesare provided with the two substrates. Meyer and Krell also showed thatlysates from cells transfected with cDNA for DG42 synthesize elevatedlevels of HA. Now that its function is known, DG42 can, therefore, bedesignated XlHAS.

[0023] There are common predicted structural features shared by all theHAS proteins, including a large central domain and clusters of 2-3transmembrane or membrane-associated domains at both the amino andcarboxyl ends of the protein. The central domain, which comprises up to˜88% of the predicted intracellular HAS protein sequences, probablycontains the catalytic regions of the enzyme. This predicted centraldomain is 264 amino acids long in spHAS (63% of the total protein) and307-328 residues long in the eukaryotic HAS members (54-56% of the totalprotein). The exact number and orientation of membrane domains and thetopological organization of extracellular and intracellular loops havenot yet been experimentally determined for any HAS.

[0024] spHAS is a HAS family member that has been purified and partiallycharacterized. Initial studies using spHAS/alkaline phosphatase fusionproteins indicate that the N terminus, C terminus, and the large centraldomain of spHAS are, in fact, inside the cell. spHAS has 6 cysteines,whereas HAS1, HAS2, and HAS3 have 13, 14 and 14 Cys residues,respectively. Two of the 6 Cys residues in spHAS are conserved andidentical in HAS1 and HAS2. Only one conserved Cys residue is found atthe same position (Cys-225 in spHAS) in all the HAS family members. Thismay be an essential Cys whose modification by sulfhydryl poisonspartially inhibits enzyme activity. The possible presence of disulfidebonds or the identification of critical Cys residues needed for any ofthe multiple HAS functions noted below has not yet been elucidated forany members of the HAS family.

[0025] In addition to the proposed unique mode of synthesis at theplasma membrane, the HAS enzyme family is highly unusual in the largenumber of functions required for the overall polymerization of HA. Atleast six discrete activities are present within the HAS enzyme: bindingsites for each of the two different sugar nucleotide precursors(UDP-GlcNAc and UDP-GlcA), two different glycosyltransferase activities,one or more binding sites that anchor the growing HA polymer to theenzyme (perhaps related to a B-X₇-B motif), and a ratchet-like transferreaction that moves the growing polymer one sugar at a time. This lateractivity is likely coincident with the stepwise advance of the polymerthrough the membrane. All of these functions, and perhaps others as yetunknown, are present in a relatively small protein ranging in size from419 (spHAS) to 588 (xHAS) amino acids.

[0026] Although all the available evidence supports the conclusion thatonly the spHAS protein is required for HA biosynthesis in bacteria or invitro, it is possible that the larger eukaryotic HAS family members arepart of multicomponent complexes. Since the eukaryotic HAS proteins are˜40% larger than spHAS, their additional protein domains could beinvolved in more elaborate functions such as intracellular traffickingand localization, regulation of enzyme activity, and mediatinginteractions with other cellular components.

[0027] The unexpected finding that there are multiple vertebrate HASgenes encoding different synthases strongly supports the emergingconsensus that HA is an important regulator of cell behavior and notsimply a structural component in tissues. Thus, in less than six months,the field moved from one known, cloned HAS (spHAS) to recognition of amultigene family that promises rapid, numerous, and exciting futureadvances in our understanding of the synthesis and biology of HA. Forexample, disclosed hereinafter are the sequences of the two HAS genes:from Pasturella multocida; and (2) Paramecium bursaria chlorella virus(PBCV-1). The presence of hyaluronan synthase in these two systems andthe purification and use of the hyaluronan synthase from these twodifferent systems indicates an ability to purify and isolate nucleicacid sequences encoding enzymatically active hyaluronan synthase in manydifferent prokaryotic and viral sources.

[0028] Group C Streptococcus equisimilis strain D181 synthesizes andsecretes hyaluronic acid (HA). Investigators have used this strain andGroup A Streptococcus pyogene strains, such as S43 and A111, to studythe biosynthesis of HA and to characterize the HA-synthesizing activityin terms of its divalent cation requirement, precursor (UDP-GlcNAc andUDP-GlcA) utilization, and optimum pH.

[0029] Traditionally, HA has been prepared commercially by isolationfrom either rooster combs or extracellular media from Streptococcalcultures. One method which has been developed for preparing HA isthrough the use of cultures of HA-producing Streptococcal bacteria. U.S.Pat. No. 4,517,295 describes such a procedure wherein HA-producingStreptococci are fermented under anaerobic conditions in a CO₂-enrichedgrowth medium. Under these conditions, HA is produced and can beextracted from the broth. It is generally felt that isolation of HA fromrooster combs is laborious and difficult, since one starts with HA in aless pure state. The advantage of isolation from rooster combs is thatthe HA produced is of higher molecular weight. However, preparation ofHA by bacterial fermentation is easier, since the HA is of higher purityto start with. Usually, however, the molecular weight of HA produced inthis way is smaller than that from rooster combs. Therefore, a techniquethat would allow the production of high molecular weight HA by bacterialfermentation would be an improvement over existing procedures.

[0030] High molecular weight HA has a wide variety of usefulapplications—ranging from cosmetics to eye surgery. Due to its potentialfor high viscosity and its high biocompatibility, HA finds particularapplication in eye surgery as a replacement for vitreous fluid. HA hasalso been used to treat racehorses for traumatic arthritis byintra-articular injections of HA, in shaving cream as a lubricant, andin a variety of cosmetic products due to its physiochemical propertiesof high viscosity and its ability to retain moisture for long periods oftime. In fact, in August of 1997 the U.S. Food and Drug Agency approvedthe use of high molecular weight HA in the treatment of severe arthritisthrough the injection of such high molecular weight HA directly into theaffected joints. In general, the higher molecular weight HA that isemployed the better. This is because HA solution viscosity increaseswith the average molecular weight of the individual HA polymer moleculesin the solution. Unfortunately, very high molecular weight HA, such asthat ranging up to 10⁷, has been difficult to obtain by currentlyavailable isolation procedures.

[0031] To address these or other difficulties, there is a need for newmethods and constructs that can be used to produce HA having one or moreimproved properties such as greater purity or ease of preparation. Inparticular, there is a need to develop methodology for the production oflarger amounts of relatively high molecular weight and relatively pureHA than is currently commercially available. There is yet another needto be able to develop methodology for the production of HA having amodified size distribution (HA_(Δsize)) as well as HA having a modifiedstructure (HA_(Δmod))

[0032] The present invention addresses one or more shortcomings in theart. Using recombinant DNA technology, a purified nucleic acid segmenthaving a coding region encoding enzymatically active seHAS is disclosedand claimed in conjunction, with methods to produce an enzymaticallyactive HA synthase, as well as methods for using the nucleic acidsegment in the preparation of recombinant cells which produce HAS andits hyaluronic acid product.

[0033] Thus, it is an object of the present invention to provide apurified nucleic acid segment having a coding region encodingenzymatically active HAS.

[0034] It is a further object of the present invention to provide arecombinant vector which includes a purified nucleic acid segment havinga coding region encoding enzymatically active HAS.

[0035] It is still a further object of the present invention to providea recombinant host cell transformed with a recombinant vector whichincludes a purified nucleic acid segment having a coding region encodingenzymatically active HAS.

[0036] It is yet another object of the present invention to provide amethod for detecting a bacterial cell that expresses HAS.

[0037] It is another object of the present invention to provide a methodfor producing high and/or low molecular weight hyaluronic acid from ahyaluronate synthase gene, such as seHAS, as well as methods forproducing HA having a modified size distribution and/or a modifiedstructure.

[0038] These and other objects of the present invention will becomeapparent in light of the attached specification, claims, and drawings.

BRIEF SUMMARY OF THE INVENTION

[0039] The present invention involves the application of recombinant DNAtechnology to solving one or more problems in the art of hyaluronic acid(HA) preparation. These problems are addressed through the isolation anduse of a nucleic acid segment having a coding region encoding theenzymatically active Streptococcus equisimilis (seHAS) hyaluronatesynthase gene, a gene responsible for HA chain biosynthesis. The seHASgene was cloned from DNA of an appropriate microbial source andengineered into useful recombinant constructs for the preparation of HAand for the preparation of large quantities of the HAS enzyme itself.

[0040] The present invention encompasses a novel gene, seHAS. Theexpression of this gene correlates with virulence of Streptococcal GroupA and Group C strains, by providing a means of escaping phagocytosis andimmune surveillance. The terms “hyaluronic acid synthase”, “hyaluronatesynthase”, “hyaluronan synthase” and “HA synthase”, are usedinterchangeably to describe an enzyme that polymerizes aglycosaminoglycan polysaccharide chain composed of alternatingglucuronic acid and N-acetylglucosamine sugars, β1,3 and β1,4 linked.The term “seHAS” describes the HAS enzyme derived from Streptococcusequisimilis.

[0041] The present invention concerns the isolation and characterizationof a hyaluronate or hyaluronic acid synthase gene, cDNA, and geneproduct (HAS), as may be used for the polymerization of glucuronic acidand N-acetylglucosamine into the glycosaminoglycan hyaluronic acid. Thepresent invention identifies the seHAS locus and discloses the nucleicacid sequence which encodes for the enzymatically active seHAS gene fromStreptococcus equisimilis. The HAS gene also provides a new probe toassess the potential of bacterial specimens to produce hyaluronic acid.

[0042] Through the application of techniques and knowledge set forthherein, those of skill in the art will be able to obtain nucleic acidsegments encoding the seHAS gene. As those of skill in the art willrecognize, in light of the present disclosure, these advantages providesignificant utility in being able to control the expression of the seHASgene and control the nature of the seHAS gene product, the seHAS enzyme,that is produced.

[0043] Accordingly, the invention is directed to the isolation of apurified nucleic acid segment which has a coding region encodingenzymatically active HAS, whether it be from prokaryotic or eukaryoticsources. This is possible because the enzyme, and indeed the gene, isone found in both eukaryotes and some prokaryotes. Eukaryotes are alsoknown to produce HA and thus have HA synthase genes that can be employedin connection with the invention.

[0044] HA synthase-encoding nucleic acid segments of the presentinvention are defined as being isolated free of total chromosomal orgenomic DNA such that they may be readily manipulated by recombinant DNAtechniques. Accordingly, as used herein, the phrase “a purified nucleicacid segment” refers to a DNA segment isolated free of unrelatedchromosomal or genomic DNA and retained in a state rendering it usefulfor the practice of recombinant techniques, such as DNA in the form of adiscrete isolated DNA fragment, or a vector (e.g., plasmid, phage orvirus) incorporating such a fragment.

[0045] A preferred embodiment of the present invention is a purifiednucleic acid segment having a coding region encoding enzymaticallyactive HAS. In particular, the purified nucleic acid segment encodes theseHAS of SEQ ID NO:2 or the purified nucleic acid segment comprises anucleotide sequence in accordance with SEQ ID NO:1.

[0046] Another embodiment of the present invention comprises a purifiednucleic acid segment having a coding region encoding enzymaticallyactive HAS and the purified nucleic acid segment is capable ofhybridizing to the nucleotide sequence of SEQ ID NO:1.

[0047] The present invention also comprises a natural or recombinantvector consisting of a plasmid, cosmid, phage, or virus vector. Therecombinant vector may also comprise a purified nucleic acid segmenthaving a coding region encoding enzymatically active HAS.

[0048] In particular, the purified nucleic acid segment encodes theseHAS of SEQ ID NO:2 or the purified nucleic acid segment comprises anucleotide sequence in accordance with SEQ ID NO:1. If the recombinantvector is a plasmid, it may further comprise an expression vector. Theexpression vector may also include a promoter operatively linked to theenzymatically active HAS coding region.

[0049] In another preferred embodiment, the present invention comprisesa recombinant host cell such as a prokaryotic cell transformed with arecombinant vector. The recombinant vector includes a purified nucleicacid segment having a coding region encoding enzymatically active HAS.In particular, the purified nucleic acid segment encodes the seHAS ofSEQ ID NO:2 or the purified nucleic acid segment comprises a nucleotidesequence in accordance with SEQ ID NO:1.

[0050] The present invention also comprises a recombinant host cell,such as an eukaryotic cell transfected with a recombinant vectorcomprising a purified nucleic acid segment having a coding regionencoding enzymatically active HAS. In particular, the purified nucleicacid segment encodes the seHAS of SEQ ID NO:2 or the purified nucleicacid segment comprises a nucleotide sequence in accordance with SEQ IDNO:1. The concept is to create a specifically modified seHAS gene thatencodes an enzymatically active HAS capable of producing a hyaluronicacid polymer having a modified structure or a modified sizedistribution.

[0051] The present invention further comprises a recombinant host cellwhich is electroporated to introduce a recombinant vector into therecombinant host cell. The recombinant vector may include a purifiednucleic acid segment having a coding region encoding enzymaticallyactive HAS. In particular, the purified nucleic acid segment encodes theseHAS of SEQ ID NO:2 or the purified nucleic acid segment comprises anucleotide sequence in accordance with SEQ ID NO:1. The enzymaticallyactive HAS may also be capable of producing a hyaluronic acid polymerhaving a modified structure or a modified size distribution.

[0052] In yet another preferred embodiment, the present inventioncomprises a recombinant host cell which is transduced with a recombinantvector which includes a purified nucleic acid segment having a codingregion encoding enzymatically active HAS. In particular, the purifiednucleic acid segment encodes the seHAS of SEQ ID NO:2 or the purifiednucleic acid segment comprises a nucleotide sequence in accordance withSEQ ID NO:1. The enzymatically active HAS is also capable of producing ahyaluronic acid polymer having a modified structure or a modified sizedistribution.

[0053] The present invention also comprises a purified composition,wherein the purified composition comprises a polypeptide having a codingregion encoding enzymatically active HAS and further having an aminoacid sequence in accordance with SEQ ID NO:2.

[0054] In another embodiment, the invention comprises a method fordetecting a DNA species, comprising the steps of: (1) obtaining a DNAsample; (2) contacting the DNA sample with a purified nucleic acidsegment in accordance with SEQ ID NO:1 ; (3) hybridizing the DNA sampleand the purified nucleic acid segment thereby forming a hybridizedcomplex; and (4) detecting the complex.

[0055] The present invention also comprises a method for detecting abacterial cell that expresses mRNA encoding seHAS, comprising the stepsof: (1) obtaining a bacterial cell sample; (2) contacting at least onenucleic acid from the bacterial cell sample with purified nucleic acidsegment in accordance with SEQ ID NO:1; (3) hybridizing the at least onenucleic acid and the purified nucleic acid segment thereby forming ahybridized complex; and (4) detecting the hybridized complex, whereinthe presence of the hybridized complex is indicative of a bacterialstrain that expresses mRNA encoding seHAS.

[0056] The present invention also comprises methods for detecting thepresence of either seHAS or spHAS in a cell. In particular, the methodcomprises using the oligonucleotides set forth in Seq. ID Nos.:3-8 asprobes. These oligonucleotides would a allow a practitioner to searchand detect the presence of seHAS or spHAS in a cell.

[0057] The present invention further comprises a method for producinghyaluronic acid, comprising the steps of: (1) introducing a purifiednucleic acid segment having a coding region encoding enzymaticallyactive HAS into a host organism, wherein the host organism containsnucleic acid segments encoding enzymes which produce UDP-GlcNAc andUDP-GlcA; (2) growing the host organism in a medium to secretehyaluronic acid; and (3) recovering the secreted hyaluronic acid.

[0058] The method may also include the step of extracting the secretedhyaluronic acid from the medium as well as the step of purifying theextracted hyaluronic acid. Furthermore, the host organism may secrete astructurally modified hyaluronic acid or a size modified hyaluronicacid.

[0059] The present invention further comprises a pharmaceuticalcomposition comprising a preselected pharmaceutical drug and aneffective amount of hyaluronic acid produced by a recombinant HAS. Thepharmaceutical composition may have a hyaluronic acid having a modifiedmolecular weight pharmaceutical composition capable of evading an immuneresponse. The modified molecular weight may also produce apharmaceutical composition capable of targeting a specific tissue orcell type within the patient having an affinity for the modifiedmolecular weight pharmaceutical composition.

[0060] The present invention also comprises a purified and isolatednucleic acid sequence encoding enzymatically active seHAS, where thenucleic acid sequence is (a) the nucleic acid sequence in accordancewith SEQ ID NO:1; (b) complementary nucleic acid sequences to thenucleic acid sequence in accordance with SEQ ID NO:1; (c) nucleic acidsequences which will hybridize to the nucleic acid in accordance withSEQ ID NO:1; and (d) nucleic acid sequences which will hybridize to thecomplementary nucleic acid sequences of SEQ ID NO:1.

[0061] The present invention further comprises a purified and isolatednucleic acid segment consisting essentially of a nucleic acid segmentencoding enzymatically active HAS.

[0062] The present invention also comprises an isolated nucleic acidsegment consisting essentially of a nucleic acid segment encoding seHAShaving a nucleic acid segment sufficiently duplicative of the nucleicacid segment in accordance of SEQ ID NO:1 to allow possession of thebiological property of encoding for an enzymatically active HAS. Thenucleic acid segment may also be a cDNA sequence.

[0063] The present invention also comprises a purified nucleic acidsegment having a coding region encoding enzymatically active HAS,wherein the purified nucleic acid segment is capable of hybridizing tothe nucleotide sequence in accordance with SEQ ID NO:1.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0064]FIG. 1 depicts that cross hybridization between seHAS and spHASgenes does not occur.

[0065]FIG. 2 figuratively depicts the relatedness of seHAS to thebacterial and eukaryotic HAS proteins.

[0066]FIG. 3 figuratively depicts evolutionary relationships among someof the known hyaluronan synthases.

[0067]FIG. 4 depicts the HA size distribution produced by variousengineered Streptococcal HAS enzymes.

[0068]FIG. 5 figuratively depicts the overexpression of recombinantseHAS and spHAS in E. coli.

[0069]FIG. 6 depicts purification of Streptococcal HA synthase.

[0070]FIG. 7 depicts a gel filtration analysis of HA synthesized byrecombinant streptococcal HAS expressed in yeast membranes.

[0071]FIG. 8 is a Western blot analysis of recombinant seHAS usingspecific antibodies.

[0072]FIG. 9 is a kinetic analysis of the HA size distributions producedby recombinant seHAS and spHAS.

[0073]FIG. 10 graphically depicts the hydropathy plots for seHAS andpredicted membrane associated regions.

[0074]FIG. 11 is a graphical model for the topologic organization ofseHAS in the membrane.

[0075]FIG. 12 is a demonstration of the synthesis of authentic HA by therecombinant seHAS.

[0076]FIG. 13 depicts the recognition of nucleic acid sequences encodingseHAS, encoding spHAS, or encoding both seHAS and spHAS using specificoligonucleotides and PCR.

[0077]FIG. 14 depicts oligonucleotides used for specific PCRhybridization.

DETAILED DESCRIPTION OF THE INVENTION

[0078] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting.

[0079] As used herein, the term “nucleic acid segment” and “DNA segment”are used interchangeably and refer to a DNA molecule which has beenisolated free of total genomic DNA of a particular species. Therefore, a“purified” DNA or nucleic acid segment as used herein, refers to a DNAsegment which contains a Hyaluronate Synthase ( “HAS”) coding sequenceyet is isolated away from, or purified free from, unrelated genomic DNA,for example, total Streptococcus equisimilis or, for example, mammalianhost genomic DNA. Included within the term “DNA segment”, are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

[0080] Similarly, a DNA segment comprising an isolated or purified seHASgene refers to a DNA segment including HAS coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case seHAS, formsthe significant part of the coding region of the DNA segment, and thatthe DNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or DNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to, or intentionally left in the segment by the hand of man.

[0081] Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HAS gene from prokaryotes such as S. pyogenes, S. equisimilis, or P.multocida. One such advantage is that, typically, eukaryotic enzymes mayrequire significant post-translational modifications that can only beachieved in a eukaryotic host. This will tend to limit the applicabilityof any eukaryotic HA synthase gene that is obtained. Moreover, those ofordinary skill in the art will likely realize additional advantages interms of time and ease of genetic manipulation where a prokaryoticenzyme gene is sought to be employed. These additional advantagesinclude (a) the ease of isolation of a prokaryotic gene because of therelatively small size of the genome and, therefore, the reduced amountof screening of the corresponding genomic library and (b) the ease ofmanipulation because the overall size of the coding region of aprokaryotic gene is significantly smaller due to the absence of introns.Furthermore, if the product of the seHAS gene (i.e., the enzyme)requires posttranslational modifications, these would best be achievedin a similar prokaryotic cellular environment (host) from which the genewas derived.

[0082] Preferably, DNA sequences in accordance with the presentinvention will further include genetic control regions which allow theexpression of the sequence in a selected recombinant host. Of course,the nature of the control region employed will generally vary dependingon the particular use (e.g., cloning host) envisioned.

[0083] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a seHAS gene, that includes within its amino acid sequence anamino acid sequence in accordance with SEQ ID NO:2. Moreover, in otherparticular embodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of anHAS gene or DNA, and in particular to an HAS gene or cDNA, correspondingto Streptococcus equisimilis HAS. For example, where the DNA segment orvector encodes a full length HAS protein, or is intended for use inexpressing the HAS protein, preferred sequences are those which areessentially as set forth in SEQ ID NO:2.

[0084] Nucleic acid segments having HA synthase activity may be isolatedby the methods described herein. The term “a sequence essentially as setforth in SEQ ID NO:2” means that the sequence substantially correspondsto a portion of SEQ ID NO:2 and has relatively few amino acids which arenot identical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:2. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein, as agene having a sequence essentially as set forth in SEQ ID NO:2, and thatis associated with the ability of prokaryotes to produce HA or ahyaluronic acid coat.

[0085] For instance, the seHAS and spHAS coding sequences areapproximately 70% identical and rich in the bases adenine (A) andthymine (T). SeHAS base content is A-26.71%, C-19.13%, G-20.81%, andT-33.33% (A/T=60%). Whereas spHAS is A-31.34%, C-16.42%, G-16.34%, andT-35.8% (A/T =67%). Those of ordinary skill in the art would besurprised that the seHAS coding sequence does not hybridize with thespHAS gene and vice versa, despite their being 70% identical. Thisunexpected inability to cross-hybridize could be due to shortinterruptions of mismatched bases throughout the open reading frames.The inability of spHAS and seHAS to cross-hybridize is shown in FIG. 1.The longest stretch of identical nucleotides common to both the seHASand the spHAS coding sequences is only 20 nucleotides. In addition, thevery A-T rich sequences will form less stable hybridization complexesthan G-C rich sequences. Another possible explanation could be thatthere are several stretches of As or Ts in both sequences that couldhybridize in a misaligned and unstable manner. This would put the seHASand spHAS gene sequences out of frame with respect to each other,thereby decreasing the probability of productive hybridization.

[0086] Because of this unique phenomena of two genes encoding proteinswhich are 70% identical not being capable of cross-hybridizing to oneanother, it is beneficial to think of the claimed nucleic acid segmentin terms of its function; i.e. a nucleic acid segment which encodesenzymatically active hyaluronate synthase. One of ordinary skill in theart would appreciate that a nucleic acid segment encoding enzymaticallyactive hyaluronate synthase may contain conserved or semi-conservedsubstitutions to the sequences set forth in SEQ ID NOS:1 and 2 and yetstill be within the scope of the invention.

[0087] In particular, the art is replete with examples of practitionersability to make structural changes to a nucleic acid segment (i.e.encoding conserved or semi-conserved amino acid substitutions) and stillpreserve its enzymatic or functional activity. See for example: (1)Risler et al. “Amino Acid Substitutions in Structurally RelatedProteins. A Pattern Recognition Approach.” J. Mol. Biol. 204:1019-1029(1988) [“. . . according to the observed exchangeability of amino acidside chains, only four groups could be delineated; (i) Ile and Val; (ii)Leu and Met, (iii) Lys, Arg, and Gln, and (iv) Tyr and Phe. ”]; (2)Niefind et al. “Amino Acid Similarity Coefficients for Protein Modelingand Sequence Alignment Derived from Main-Chain Folding Anoles.” J. Mol.Biol. 219:481-497 (1991) [similarity parameters allow amino acidsubstitutions to be designed]; and (3) Overington et al.“Environment-Specific Amino Acid Substitution Tables: Tertiary Templatesand Prediction of Protein Folds,” Protein Science 1:216-226 (1992)[“Analysis of the pattern of observed substitutions as a function oflocal environment shows that there are distinct patterns . . .”Compatible changes can be made.]

[0088] These references and countless others, indicate that one ofordinary skill in the art, given a nucleic acid sequence, could makesubstitutions and changes to the nucleic acid sequence without changingits functionality. Also, a substituted nucleic acid segment may behighly identical and retain its enzymatic activity with regard to itsunadulterated parent, and yet still fail to hybridize thereto.

[0089] The invention discloses nucleic acid segments encodingenzymatically active hyaluronate synthase—seHAS and spHAS. AlthoughseHAS and spHAS are 70% identical and both encode enzymatically activehyaluronate synthase, they do not cross hybridize. Thus, one of ordinaryskill in the art would appreciate that substitutions can be made to theseHAS nucleic acid segment listed in SEQ ID NO:1 without deviatingoutside the scope and claims of the present invention. Standardized andaccepted functionally equivalent amino acid substitutions are presentedin Table I. TABLE I Conservative and Semi- Amino Acid Group ConservativeSubstitutions NonPolar R Groups Alanine, Valine, Leucine, Isoleucine,Proline, Methionine, Phenylalanine, Tryptophan Polar, but uncharged, RGlycine, Serine, Threonine, Groups Cysteine, Asparagine, GlutamineNegatively Charged R Groups Aspartic Acid, Glutamic Acid PositivelyCharged R Groups Lysine, Arginine, Histidine

[0090] Another preferred embodiment of the present invention is apurified nucleic acid segment that encodes a protein in accordance withSEQ ID NO:2, further defined as a recombinant vector. As used herein,the term “recombinant vector” refers to a vector that has been modifiedto contain a nucleic acid segment that encodes an HAS protein, orfragment thereof. The recombinant vector may be further defined as anexpression vector comprising a promoter operatively linked to said HASencoding nucleic acid segment.

[0091] A further preferred embodiment of the present invention is a hostcell, made recombinant with a recombinant vector comprising an HAS gene.The preferred recombinant host cell may be a prokaryotic cell. Inanother embodiment, the recombinant host cell is a eukaryotic cell. Asused herein, the term “engineered” or “recombinant” cell is intended torefer to a cell into which a recombinant gene, such as a gene encodingHAS, has been introduced. Therefore, engineered cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced gene. Engineered cells are thus cells having agene or genes introduced through the hand of man. Recombinantlyintroduced genes will either be in the form of a cDNA gene, a copy of agenomic gene, or will include genes positioned adjacent to a promoternot naturally associated with the particular introduced gene.

[0092] Where one desires to use a host other than Streptococcus, as maybe used to produce recombinant HA synthase, it may be advantageous toemploy a prokaryotic system such as E. coli, B. subtilis, Lactococcussp., or even eukaryotic systems such as yeast or Chinese hamster ovary,African green monkey kidney cells, VERO cells, or the like. Of course,where this is undertaken it will generally be desirable to bring the HAsynthase gene under the control of sequences which are functional in theselected alternative host. The appropriate DNA control sequences, aswell as their construction and use, are generally well known in the artas discussed in more detail hereinbelow.

[0093] In preferred embodiments, the HA synthase-encoding DNA segmentsfurther include DNA sequences, known in the art functionally as originsof replication or “replicons”, which allow replication of contiguoussequences by the particular host. Such origins allow the preparation ofextrachromosomally localized and replicating chimeric segments orplasmids, to which HA synthase DNA sequences are ligated. In morepreferred instances, the employed origin is one capable of replicationin bacterial hosts suitable for biotechnology applications. However, formore versatility of cloned DNA segments, it may be desirable toalternatively or even additionally employ origins recognized by otherhost systems whose use is contemplated (such as in a shuttle vector).

[0094] The isolation and use of other replication origins such as theSV40, polyoma or bovine papilloma virus origins, which may be employedfor cloning or expression in a number of higher organisms, are wellknown to those of ordinary skill in the art. In certain embodiments, theinvention may thus be defined in terms of a recombinant transformationvector which includes the HA synthase coding gene sequence together withan appropriate replication origin and under the control of selectedcontrol regions.

[0095] Thus, it will be appreciated by those of skill in the art thatother means may be used to obtain the HAS gene or cDNA, in light of thepresent disclosure. For example, polymerase chain reaction or RT-PCRproduced DNA fragments may be obtained which contain full complements ofgenes or cDNAs from a number of sources, including other strains ofStreptococcus or from eukaryotic sources, such as cDNA libraries.Virtually any molecular cloning approach may be employed for thegeneration of DNA fragments in accordance with the present invention.Thus, the only limitation generally on the particular method employedfor DNA isolation is that the isolated nucleic acids should encode abiologically functional equivalent HA synthase.

[0096] Once the DNA has been isolated it is ligated together with aselected vector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovinepapilloma virus and retroviruses. However, it is believed thatparticular advantages will ultimately be realized where vectors capableof replication in both Lactococcus or Bacillus strains and E. coli areemployed.

[0097] Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti or the pAT19 vector of Trieu-Cuot, et al., allow one to performclonal colony selection in an easily manipulated host such as E. coli,followed by subsequent transfer back into a food grade Lactococcus orBacillus strain for production of HA. These are benign and well studiedorganisms used in the production of certain foods and biotechnologyproducts. These are advantageous in that one can augment the Lactococcusor Bacillus strain's ability to synthesize HA through gene dosaging(i.e., providing extra copies of the HA synthase gene by amplification)and/or inclusion of additional genes to increase the availability of HAprecursors. The inherent ability of a bacterium to synthesize HA canalso be augmented through the formation of extra copies, oramplification, of the plasmid that carries the HA synthase gene. Thisamplification can account for up to a 10-fold increase in plasmid copynumber and, therefore, the HA synthase gene copy number.

[0098] Another procedure that would further augment HA synthase genecopy number is the insertion of multiple copies of the gene into theplasmid. Another technique would include integrating the HAS gene intochromosomal DNA. This extra amplification would be especially feasible,since the bacterial HA synthase gene size is small. In some scenarios,the chromosomal DNA-ligated vector is employed to transfect the hostthat is selected for clonal screening purposes such as E. coli, throughthe use of a vector that is capable of expressing the inserted DNA inthe chosen host.

[0099] Where a eukaryotic source such as dermal or synovial fibroblastsor rooster comb cells is employed, one will desire to proceed initiallyby preparing a cDNA library. This is carried out first by isolation ofmRNA from the above cells, followed by preparation of double strandedcDNA using an enzyme with reverse transcriptase activity and ligationwith the selected vector. Numerous possibilities are available and knownin the art for the preparation of the double stranded cDNA, and all suchtechniques are believed to be applicable. A preferred technique involvesreverse transcription. Once a population of double stranded cDNAs isobtained, a cDNA library is prepared in the selected host by acceptedtechniques, such as by ligation into the appropriate vector andamplification in the appropriate host. Due to the high number of clonesthat are obtained, and the relative ease of screening large numbers ofclones by the techniques set forth herein, one may desire to employphage expression vectors, such as λgt11, λgt12, λGem11, and/or λZAP forthe cloning and expression screening of cDNA clones.

[0100] In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1” is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:1. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table I, and also refers tocodons that encode biologically equivalent amino acids.

[0101] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ nucleic acid sequences, and yet stillbe essentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzyme activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes. In particular, the amino acidsequence of the HAS gene in eukaryotes appears to be 40% larger thanthat found in prokaryotes.

[0102] Allowing for the degeneracy of the genetic code as well asconserved and semi-conserved substitutions, sequences which have betweenabout 40% and about 80%; or more preferably, between about 80% and about90%; or even more preferably, between about 90% and about 99%; ofnucleotides which are identical to the nucleotides of SEQ ID NO:1 willbe sequences which are “essentially as set forth in SEQ ID NO:1”.Sequences which are essentially the same as those set forth in SEQ IDNO:1 may also be functionally defined as sequences which are capable ofhybridizing to a nucleic acid segment containing the complement of SEQID NO:1 under standard or less stringent hybridizing conditions.Suitable standard hybridization conditions will be well known to thoseof skill in the art and are clearly set forth herein.

[0103] The term “standard hybridization conditions” as used herein, isused to describe those conditions under which substantiallycomplementary nucleic acid segments will form standard Watson-Crickbase-pairing. A number of factors are known that determine thespecificity of binding or hybridization, such as pH, temperature, saltconcentration, the presence of agents, such as formamide and dimethylsulfoxide, the length of the segments that are hybridizing, and thelike. When it is contemplated that shorter nucleic acid segments will beused for hybridization, for example fragments between about 14 and about100 nucleotides, salt and temperature preferred conditions forhybridization will include 1.2-1.8×HPB at 40-50° C.

[0104] Naturally, the present invention also encompasses DNA segmentswhich are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NO:1. Nucleic acid sequences which are“complementary” are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. As used herein, theterm “complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1.

[0105] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that theiroverall length may vary considerably. It is therefore contemplated thata nucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

[0106] Naturally, it will also be understood that this invention is notlimited to the particular nucleic acid and amino acid sequences of SEQID NO:1 and 2. Recombinant vectors and isolated DNA segments maytherefore variously include the HAS coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, or they may encode larger polypeptides which neverthelessinclude HAS-coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acidssequences.

[0107] For instance, we have found, characterized, and purifiedhyaluronate synthase in two other systems: (a) the gram-negativebacteria Pasturella multocida (SEQ ID NO:9); and (2) chlorella virusPBCV-1 (SEQ ID NOS:7 and 8). The presence of hyaluronan synthase inthese two systems and our ability to purify and use the hyaluronansynthase from these two different systems indicates our ability topurify and isolate nucleic acid sequences encoding enzymatically activehyaluronan synthase.

[0108] The capsule of Carter Type A P. multocida (SEQ ID NO:9) was longsuspected of containing hyaluronic acid-HA. Characterization of the HAsynthase of P. multocida led to interesting enzymological differencesbetween it and the seHAS and spHAS proteins.

[0109]P. multocida cells produce a readily visible extracellular HAcapsule, and since the two streptococcal HASs are membrane proteins,membrane preparations of the fowl cholera pathogen were tested. In earlytrials, crude membrane fractions derived from ultrasonication alonepossessed very low levels of UDP-GlcNAc-dependent UDP-[¹⁴C]GlcAincorporation into HA[˜0.2 pmol of GlcA transfer (μg of proteins) ⁻¹h⁻¹]when assayed under conditions similar to those for measuringstreptococcal HAS activity. The enzyme from E. coli with the recombinanthasA plasmid was also recalcitrant to isolation at first. These resultswere in contrast to the easily detectable amounts obtained fromStreptococcus by similar methods.

[0110] An alternative preparation protocol using ice-cold lysozymetreatment in the presence of protease inhibitors in conjunction withultrasonication allowed the substantial recovery of HAS activity fromboth species of Gram-negative bacteria. Specific activities for HAS of5-10 pmol of GlcA transferred (μg of protein)⁻¹h⁻¹ were routinelyobtained for crude membranes of wild-type P. multocida with the newmethod. In the absence of UDP-GlcNAc, virtually no radioactivity (<1% ofidentical assay with both sugar precursors) from UDP-[¹⁴C]GlcA wasincorporated into higher molecular weight material. Membranes preparedfrom the acapsular mutant, TnA, possessed no detectable HAS activitywhen supplemented with both sugar nucleotide precursors (data notshown). Gel-filtration analysis using a Sephacryl S-200 column indicatesthat the molecular mass of the majority of the ¹⁴C-labeled productsynthesized in vitro is ≧8×10⁴ Da since the material elutes in the voidvolumes, such a value corresponds to a HA molecule composed of at least400 monomers. This product is sensitive to Streptomyces hyaluronidasedigestion but resistant to protease treatment.

[0111] The parameters of the HAS assay were varied to maximizeincorporation of UDP-sugars into polysaccharide by P. multocidamembranes. Streptococcal spHAS requires Mg²+ and therefore this metalion was included in the initial assays of P. multocida membranes. The P.multocida HAS (pmHAS) was relatively active from pH 6.5 to 8.6 inTris-type buffers with an optimum at pH 7. The HAS activity was linearwith respect to the incubation time at neutral pH for at least 1 h. ThepmHAS was apparently less active at higher ionic strengths because theaddition of 100 mM NaCl to the reaction containing 50 mM Tris, pH 7, and20 mM MgCl₂ reduced sugar incorporation by ˜50%.

[0112] The metal ion specificity of the pmHAS was assessed at pH 7.Under metal-free conditions in the presence of EDTA, no incorporation ofradiolabeled precursor into polysaccharide was detectable (<0.5% ofmaximal signal). Mn²⁺ gave the highest incorporation rates at the lowestion concentrations for the tested metals (Mg, Mn, Co, Cu, and Ni). Mg²⁺gave about 50% of the Mn²⁺ stimulation but at 10-fold higherconcentrations. Co²⁺ or Ni²⁺ at 10 mM supported lower levels of activity(20% or 9%, respectively, of 1 mM Mn²⁺ assays), but membranes suppliedwith 10 mM Cu²⁺ were inactive. Indeed, mixing 10 mM Cu²⁺ and 20 mM2+Mg2+ with the membrane preparation resulted in almost no incorporationof label into polysaccharide (<0.8% of Mg only value).

[0113] Initial characterization of the pmHAS was performed in thepresence of Mg²⁺. The binding affinity of the enzyme for its sugarnucleotide precursors was assessed by measuring the apparent K_(M)value. Incorporation of [¹⁴C] GlcA or [³H] GlcNAc into polysaccharidewas monitored at varied concentrations of UDP-GlcNAc or UDP-GlcA,respectively. In Mg²⁺-containing buffers, the apparent K_(M) values of˜20 μM for UDP-GlcA and ˜75 μM for UDP-GlcNAc were determined utilizingHanes-Woolf plots ([S]/v versus [S]) of the titration data. The V_(max)values for both sugars were the same because the slopes, correspondingto 1/V_(max), of the Hanes-Woolf plots were equivalent. In comparison toresults from assays with Mg²⁺, the K_(M) value for UDP-GlcNAc wasincreased by about 25-50% to ˜10⁵ μM and the V_(max) increased by afactor of 2-3-fold in the presence of Mn²⁺.

[0114] The HA synthase enzymes from either P. multocida, S. equisimilis,or S. pyogenes utilizes UDP-sugars, but they possess somewhat differentkinetic optima with respect to pH and metal ion dependence and K_(M)values. The enzymes are most active at pH 7; however, the pmHASreportedly displays more activity at slightly acidic pH and isrelatively inactive above pH 7.4. The pmHAS utilizes Mn²⁺ moreefficiently than Mg²⁺ under the in vitro assay conditions, but theidentity of the physiological metal cofactor in the bacterial cell isunknown. In comparison, in previous studies with the streptococcalenzyme, Mg²⁺ was much better than Mn²⁺ but the albeit smaller effect ofMn²⁺ was maximal at ˜10-fold lower concentrations than the optimal Mg²⁺concentration. The pmHAS apparently binds the UDP-sugars more tightlythan spHAS. The measured K_(M) values for the pmHAS in crude membranesare about 2-3-fold lower for each substrate than those obtained from theHAS found in streptococcal membranes:50 or 39 μM for UDP-GlcA and 500 or150 μM for UDP-GlcNAc, respectively.

[0115] By kinetic analyses, the V_(max) of the pmHAS was 2-3-fold higherin the presence of Mn²⁺ than Mg²⁺, but the UDP-GlcNAc K_(M) value wasincreased slightly in assays with the former ion. This observation ofapparent lowered affinity suggests that the increased polymerizationrate was not due to better binding of the Mn²⁺ ion/sugar nucleotidecomplex to the enzyme active site(s). Therefore, it is possible thatMn²⁺ enhances some other reaction step, alters another site/structure ofthe enzyme, or modifies the phospholipid membrane environment. The genesequence and the protein sequence of pmHAS are shown in SEQ ID NO:9.

[0116] Chlorella virus PBCV-l encodes a functional glycosyltransferasethat can synthesize a polysaccharide, hyaluronan [hyaluronic acid, HA].This finding is contrary to the general observation that viruses either:(a) utilize host cell glycosyltransferases to create new carbohydratestructures, or (b) accumulate host cell glycoconjugates during virionmaturation. Furthermore, HA has been generally regarded as restricted toanimals and a few of their virulent bacterial pathogens. Though manyplant carbohydrates have been characterized, neither HA nor a relatedanalog has previously been detected in cells of plants or protists.

[0117] The vertebrate HAS enzymes (DG42, HAS1, HAS2, HAS3) andstreptococcal HasA enzymes (spHAS and seHAS) have several regions ofsequence similarity. While sequencing the double-stranded DNA genome ofvirus PBCV-1 [Paramecium bursaria chlorella virus], an ORF [open readingframe], A98R (Accession #442580), encoding a 567 residue protein with 28to 33% amino acid identity to the various HASs was discovered. Thisprotein is designated cvHAS (chlorella virus HA synthase). The genesequence encoding PBCV-1 and its protein sequence are shown in SEQ IDNOS:7 and 8.

[0118] PBCV-1 is the prototype of a family (Phycodnarviridae) of large(175-190 nm diameter) polyhedral, plaque-forming viruses that replicatein certain unicellular, eukaryotic chlorella-like green algae. PBCV-1virions contain at least 50 different proteins and a lipid componentlocated inside the outer glycoprotein capsid. The PBCV-1 genome is alinear, nonpermuted 330-kb dsDNA molecule with covalently closed hairpinends.

[0119] Based on its deduced amino acid sequence, the A98R gene productshould be an integral membrane protein. To test this hypothesis,recombinant A98R was produced in Escherichia coli and the membranefraction was assayed for HAS activity. UDP-GlcA and UDP-GlcNAc wereincorporated into the polysaccharide by the membrane fraction derivedfrom cells containing the A98R gene on a plasmid, pCVHAS, (averagespecific activity 2.5 pmoles GlcA transfer/μg protein/min) but not bysamples from control cells (<0.001 pmoles GlcA transfer/μg protein/min).No activity was detected in the soluble fraction of cells transformedwith pCVHAS. UDP-GlcA and UDP-GlcNAc were simultaneously required forpolymerization. The activity was optimal in Hepes buffer at pH 7.2 inthe presence of 10 mM MnCl₂, whereas no activity was detected if themetal ion was omitted. Mg²⁺ and Co²⁺ were ˜20% as effective as Mn²⁺ atsimilar concentrations. The pmHAS has a similar metal requirement, butother HASs prefer Mg2+.

[0120] The recombinant A98R enzyme synthesized a polysaccharide with anaverage molecular weight of 3-6×10⁶ Da which is smaller than that of theHA synthesized by recombinant spHAS or DG42×lHAS in vitro (˜107 Da and˜5-8×10⁶ Da, respectively; 13,15). The polysaccharide was completelydegraded by Streptomyces hyaluroniticus HA lyase, an enzyme thatdepolymerizes HA, but not structurally related glycosaminoglycans suchas heparin and chondroitin.

[0121] PBCV-1 infected chlorella cells were examined for A98R geneexpression. A ˜1,700-nucleotide A98R transcript appeared at ˜15 minpost-infection and disappeared by 60 min after infection indicating thatA98R is an early gene. Consequently, membrane fractions from uninfectedand PBCV-1 infected chlorella cells were assayed at 50 and 90 minpost-infection for HAS activity. Infected cells, but not uninfectedcells, had activity. Like the bacterially derived recombinant A98Renzyme, radiolabel incorporation from UDP-[¹⁴C]GlcA into polysaccharidedepended on both Mn²⁺ and UDP-GlcNAc. This radiolabeled produce was alsodegraded by HA lyase. Disrupted PBCV-1 virions had no HAS activity.

[0122] PBCV-1 infected chlorella cells were analyzed for HApolysaccharide using a highly specific ¹²⁵I-labeled HA-binding protein.Extracts from cells at 50 and 90 min post-infection containedsubstantial amounts of HA, but not extracts from uninfected algae ordisrupted PBCV-1 virions. The labeled HA-binding protein also interactedwith intact infected cells at 50 and 90 min post-infection, but nothealthy cells. Therefore, a considerable portion of the newlysynthesized HA polysaccharide was immobilized at the outer cell surfaceof the infected algae. The extracellular HA does not play any obviousrole in the interaction between the virus and its algal host becauseneither plaque size nor plaque number was altered by including eithertesticular hyaluronidase (465 units/ml) or free HA polysaccharide (100μg/ml) in the top agar of the PBCV-1 plaque assay.

[0123] The PBCV-1 genome also has additional genes that encode for anUDP-Glc dehydrogenase (UDP-Glc DH) and a glutamine:fructose-6-phosphateaminotransferase (GFAT). UDP-Glc DH converts UDP-Glc into UDP-GlcA, arequired precursor for HA biosynthesis. GFAT convertsfructose-6-phosphate into glucosamine-6-phosphate, an intermediate inthe UDP-GlcNAc metabolic pathway. Both of these PBCV-1 genes, like theA98R HAS, are expressed early in infection and encode enzymaticallyactive proteins. The presence of multiple enzymes in the HA biosynthesispathway indicates that HA production must serve an important function inthe life cycle of the chlorella viruses.

[0124] HA synthases of Streptococcus, vertebrates, and PBCV-1 possessmany motifs of 2 to 4 residues that occur in the same relative order.These conserved motifs probably reflect domains crucial for HAbiosynthesis as shown in FIG. 2. The protein sequences of Group C seHAS,Group A spHAS, murine HAS1, HAS2, HAS3, and frog HAS are shown alignedin FIG. 2. The alignment of FIG. 2 was accomplished using the DNAsIsmultiple alignment program. Residues in seHAS identical in other knownHAS family members (including human HAS1 and 2, not shown) are denotedby shading and asterisks. The amino acids indicated by dots areconserved in all members of the larger β-glycosyl transferase family.The diamond symbol indicates the highly conserved cysteine residue thatmay be critical for enzyme activity. The approximate mid-points ofpredicted membrane domains MD1 through MD7 are indicated with arrows. X1indicates Xeopus laevis, and MM denotes Mus musculis.

[0125] Regions of similarity between HASs and other enzymes thatsynthesize β-linked polysaccharides from UDP-sugar precursors are alsobeing discovered as more glycosyltransferases are sequenced. Examplesinclude bacterial cellulose synthase, fungal and bacterial chitinsynthases, and the various HASs. The significance of these similarstructural motifs will become more apparent as the three-dimensionalstructures of glycosyltransferases accumulate.

[0126]FIG. 3 depicts the evolutionary relationships among the knownhyaluronan synthase. The phylogenetic tree of FIG. 3 was generated bythe Higgins-Sharp algorithm using the DNAsis multiple alignment program.The calculated matching percentages are indicated at each branch of thedendrogram.

[0127] The DNA segments of the present invention encompass biologicallyfunctional equivalent HAS proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the enzyme activity or to antigenicity of the HASprotein or to test HAS mutants in order to examine HA synthase activityat the molecular level.

[0128] Also, specific changes to the HAS coding sequence can result inthe production of HA having a modified size distribution or structuralconfiguration. One of ordinary skill in the art would appreciate thatthe HAS coding sequence can be manipulated in a manner to produce analtered hyaluronate synthase which in turn is capable of producinghyaluronic acid having differing polymer sizes and/or functionalcapabilities. For example, the HAS coding sequence may be altered insuch a manner that the hyaluronate synthase has an altered sugarsubstrate specificity so that the hyaluronate synthase creates a newhyaluronic acid-like polymer incorporating a different structure such asa previously unincorporated sugar or sugar derivative. This newlyincorporated sugar could result in a modified hyaluronic acid havingdifferent functional properties, a hyaluronic acid having a smaller orlarger polymer size/molecular weight, or both. As will be appreciated byone of ordinary skill in the art given the HAS coding sequences, changesand/or substitutions can be made to the HAS coding sequence such thatthese desired property and/or size modifications can be accomplished.Table II lists sugar nucleotide specificity and magnesium ionrequirement of recombinant seHAS. TABLE II Sugar nucleotide specificityand Magnesium ion requirement of recombinant seHAS HA Synthesis* SecondSugar nucleotide UDP-[¹⁴C] GlcA UDP-[³H]GlcNAc present (μM) dpm (%) dpm(%) None 90 (2.1%) 8 (1.2%) UDP-GlcNAc (300) 4134 (100%) — UDP-GlcA(120) — 635 (100%) UDP-Glc (160) 81 (1.9%) 10 (1.5%) UDP-GalNAc (280) 74(1.7%) 19 (2.9%) UDP-GalA (150) 58 (1.4%) 19 (2.9%) UDP-GlcNAc + EDTA 31(0.7%) — UDP-GlcA + EDTA — 22 (3.4%)

[0129] The term “modified structure” as used herein denotes a hyaluronicacid polymer containing a sugar or derivative not normally found in thenaturally occurring HA polysaccharide. The term “modified sizedistribution” refer to the synthesis of hyaluronic acid molecules of asize distribution not normally found with the native enzyme; theengineered size could be much smaller or larger than normal.

[0130] Various hyaluronic acid products of differing size haveapplication in the areas of drug delivery and the generation of anenzyme of altered structure can be combined with a hyaluronic acid ofdiffering size. Applications in angiogenesis and wound healing arepotentially large if hyaluronic acid polymers of about 20monosaccharides can be made in good quantities. Another particularapplication for small hyaluronic acid oligosaccharides is in thestabilization of recombinant human proteins used for medical purposes. Amajor problem with such proteins is their clearance from the blood and ashort biological half life. One present solution to this problem is tocouple a small molecule shield that prevents the protein from beingcleared from the circulation too rapidly. Very small molecular weighthyaluronic acid is well suited for this role and would be nonimmunogenicand biocompatible. Larger molecular weight hyaluronic acid attached to adrug or protein may be used to target the reticuloendothelial cellsystem which has endocytic receptors for hyaluronic acid.

[0131] One of ordinary skill in the art given this disclosure wouldappreciate that there are several ways in which the size distribution ofthe hyaluronic acid polymer made by the hyaluronate synthase could beregulated to give different sizes. First, the kinetic control of productsize can be altered by decreasing temperature, decreasing time of enzymeaction and by decreasing the concentration of one or both sugarnucleotide substrates. Decreasing any or all of these variables willgive lower amounts and smaller sizes of hyaluronic acid product. Thedisadvantages of these approaches are that the yield of product willalso be decreased and it may be difficult to achieve reproducibilityfrom clay to day or batch to batch.

[0132] Secondly, the alteration of the intrinsic ability of the enzymeto synthesize a large hyaluronic acid product. Changes to the proteincan be engineered by recombinant DNA technology, including substitution,deletion and addition of specific amino acids (or even the introductionof prosthetic groups through metabolic processing). Such changes thatresult in an intrinsically slower enzyme could then allow morereproducible control of hyaluronic acid size by kinetic means. The finalhyaluronic acid size distribution is determined by certaincharacteristics of the enzyme, that rely on particular amino acids inthe sequence. Among the 20% of residues absolutely conserved between thestreptococcal enzymes and the eukaryotic hyaluronate synthases, there isa set of amino acids at unique positions that control or greatlyinfluence the size of the hyaluronic acid polymer that the enzyme canmake. Specific changes in any of these residues can produce a modifiedHAS that produces an HA product having a modified size distribution.Engineered changes to seHAS, spHAS, pmHAS, or cvHAS that decrease theintrinsic size of the hyaluronic acid that the enzyme can make beforethe hyaluronic acid is released, will provide powerful means to producehyaluronic acid product of smaller or potentially larger size than thenative enzyme.

[0133] Finally, larger molecular weight hyaluronic acid made be degradedwith specific hyaluronidases to make lower molecular weight hyaluronicacid. This practice, however, is very difficult to achievereproducibility and one must meticulously repurify the hyaluronic acidto remove the hyaluronidase and unwanted digestion products.

[0134] As shown in FIG. 4, hyaluronan synthase can be engineered toproduce hyaluronic acid polymers of different size, in particularsmaller, than the normal wildtype enzyme. The figure shows thedistribution of HA sizes (in millions of Daltons, a measure of molecularweight) for a series of spHAS enzymes, each of which was engineered bysite directed mutagenesis to have a single amino acid change from thenative enzyme. Each has a different Cysteine residue replaced withAlanine. The cluster of five curves with open symbols represent thefollowing spHAS proteins: wildtype, C124A, C261A, C366A, and C402A. Thefilled circles represent the poorly expressed C225A protein which isonly partially active.

[0135] The filled triangles is the C280A spHAS protein, which is foundto synthesize a much smaller range of HA polymers than the normal enzymeor the other variants shown. This reduction to practice shows that it isfeasible to engineer the hyaluronate synthase enzyme to synthesize adesired range of HA product sizes. The seHAS, pmHAS, and cvHAS genesencoding hyaluronate synthase can also be manipulated by site directedmutagenesis to produce an enzyme which synthesizes a desired range of HAproduct sizes.

[0136] Structurally modified hyaluronic acid is no differentconceptually than altering the size distribution of the hyaluronic acidproduct by changing particular amino acids in the desired HAS or thespHAS. Derivatives of UDP-GlcNAc, in which the N-acetyl group is missingUDP-GlcN or replaced with another chemically useful group, are expectedto be particularly useful. The strong substrate specificity must rely ona particular subset of amino acids among the 20% that are conserved.Specific changes to one or more of these residues creates a functionalsynthase that interacts less specifically with one or more of thesubstrates than the native enzyme. This altered enzyme could thenutilize alternate natural or special sugar nucleotides to incorporatesugar derivatives designed to allow different chemistries to be employedfor the following purposes: (i) covalently coupling specific drugs,proteins, or toxins to the structurally modified hyaluronic acid forgeneral or targeted drug delivery, radiological procedures, etc. (ii)covalently cross linking the hyaluronic acid itself or to other supportsto achieve a gel, or other three dimensional biomaterial with strongerphysical properties, and (iii) covalently linking hyaluronic acid to asurface to create a biocompatible film or monolayer.

[0137] Bacteria can also be engineered to produce hyaluronic acid. Forinstance, we have created strains of B. subtilis containing the spHASgene, as well as the gene for one of the sugar nucleotide precursors. Wechose this bacteria since it is frequently used in the biotech industryfor the production of products for human use. These bacteria wereintended as first generation prototypes for the generation of abacterium able to produce hyaluronic acid in larger amounts thanpresently available using a wild type natural strain. We put in multiplecopies of these genes. For example, three Bacillus subtilis strains wereconstructed to contain one or both of the Streptococcus pyogenes genesfor hyaluronan synthase (spHAS) and UDP-glucose dehydrogenase, theresults of which are shown in Table II-B. Based on a sensitivecommercial radiometric assay to detect and quantitate HA, it wasdetermined that the strain with both genes (strain #3) makes andsecretes HA into the medium. The parent strain or the strain with justthe dehydrogenase gene (strain #1) does not make HA. Strain #2, whichcontains just the spHAS gene alone makes HA, but only 10% of what strain#3 makes. Agarose gel electrophoresis showed that the HA secreted intothe medium by strain #3 is very high molecular weight. TABLE II-B CellStrain Strain with density Number Cells Medium (*) genes (A₆₀₀) (μg HAper ml of culture) 1 0 0 hasB 4.8 2 4 35 SpHAS 3.9 3 =>10 >250 SpHAS +3.2 hasB

[0138] These experiments used the streptococcal promoters normally foundwith these genes to drive protein expression. It is expected that theconstruction of strains with the spHAS or seHAS reading frame undercontrol of a B. subtilis promoter would yield even more superiorresults. The vector used is a Gram positive/E. Coli shuttle vector thathas a medium copy number in B. subtilis and a gene for erythromycinresistance (enabling resistence to 8 μg/ml in B. subtilis or 175 μg/mlin E. coli). The B. subtilis host strain used is 1A1 from BGSC, whichhas a tryptophan requirement but otherwise is wildtype, and cansporulate. Cell growth and HA production was in Spizizens Minimal Mediaplus tryptophan, glucose, trace elements and erthromycin (8 μg/ml).Growth was at 32 degrees Celsius with vigorous agitation until themedium was exhausted (˜36 hours).

[0139] This demonstrates that these bioengineered cells, which would notnormally make hyaluronic acid, became competent to do so when they aretransformed with the spHAS gene. The seHAS would also be capable ofbeing introduced into a non-hyaluronic acid producing bacteria to createa bioengineered bacterial strain capable of producing hyaluronic acid.

[0140] A preferred embodiment of the present invention is a purifiedcomposition comprising a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2. The term “purified” as used herein, isintended to refer to an HAS protein composition, wherein the HAS proteinor appropriately modified HAS protein (e.g. containing a [HIS]₆ tail) ispurified to any degree relative to its naturally-obtainable state, i.e.,in this case, relative to its purity within a prokaryotic cell extract.HAS protein may be isolated from Streptococcus, Pasturella, chlorellavirus, patient specimens, recombinant cells, infected tissues, isolatedsubpopulation of tissues that contain high levels of hyaluronate in theextracellular matrix, and the like, as will be known to those of skillin the art, in light of the present disclosure. For instance, therecombinant seHAS or spHAS protein makes up approximately 10% of thetotal membrane protein of E. coli. A purified HAS protein compositiontherefore also refers to a polypeptide having the amino acid sequence ofSEQ ID NO:2, free from the environment in which it may naturally occur(FIG. 5).

[0141] Turning to the expression of the seHAS gene whether from genomicDNA, or a cDNA, one may proceed to prepare an expression system for therecombinant preparation of the HAS protein. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression.

[0142] HAS may be successfully expressed in eukaryotic expressionsystems, however, the inventors aver that bacterial expression systemscan be used for the preparation of HAS for all purposes. It is believedthat bacterial expression will ultimately have advantages overeukaryotic expression in terms of ease of use, cost of production, andquantity of material obtained thereby.

[0143] The purification of streptococcal hyaluronan synthase (seHAS andspHAS) is shown in Table III and FIG. 6. Fractions from various stagesof the purification scheme were analyzed by SDS-PAGE on a 12.5% gel,which was then stained with Coomassie Brilliant Blue R-250. Lanes:molecular weight markers; 1, whole E. coli membranes containing therecombinant seHAS-H6; 2, insoluble fraction after detergentsolubilization of membranes; 3, detergent solubilized fraction; 4,flow-through from the Ni-NTA chromatography resin; 5-9, five successivewashes of the column (two column volumes each); 10, the eluted pure HAsynthase which is a single band. TABLE III Specific Total Total ActivityActivity Protein (mmol/ug/ (nmol Yield Purification Step (ug) hr.UDP-GlcA) (%) (-fold) Membranes 3690 1.0 3649 100 1.0 Extract 2128 2.24725 129 2.2 Affinity 39 13 500 14 13.1 Column

[0144] It is proposed that transformation of host cells with DNAsegments encoding HAS will provide a convenient means for obtaining aHAS protein. It is also proposed that cDNA, genomic sequences, andcombinations thereof, are suitable for eukaryotic expression, as thehost cell will, of course, process the genomic transcripts to yieldfunctional mRNA for translation into protein.

[0145] Another embodiment of the present invention is a method ofpreparing a protein composition comprising growing a recombinant hostcell comprising a vector that encodes a protein which includes an aminoacid sequence in accordance with SEQ ID NO:2 or functionally similarwith conserved or semi-conserved amino acid changes. The host cell willbe grown under conditions permitting nucleic acid expression and proteinproduction followed by recovery of the protein so produced. Theproduction of HAS and ultimately HA, including the host cell, conditionspermitting nucleic acid expression, protein production and recovery willbe known to those of skill in the art in light of the present disclosureof the seHAS gene, and the seHAS gene protein product HAS, and by themethods described herein.

[0146] Preferred hosts for the expression of hyaluronic acid areprokaryotes, such as S. equisimilis, and other suitable members of theStreptococcus species. However, it is also known that HA may besynthesized by heterologous host cells expressing recombinant HAsynthase, such as species members of the Bacillus, Enterococcus, or evenEscherichia genus. A most preferred host for expression of the HAsynthase of the present invention is a bacteria transformed with the HASgene of the present invention, such as Lactococcus species, Bacillussubtilis or E. coli.

[0147] It is similarly believed that almost any eukaryotic expressionsystem may be utilized for the expression of HAS e.g.,baculovirus-based, glutamine synthase-based, dihydrofolatereductase-based systems, SV-40 based, adenovirus-based,cytomegalovirus-based, yeast-based, and the like, could be employed. Forexpression in this manner, one would position the coding sequencesadjacent to and under the control of the promoter. It is understood inthe art that to bring a coding sequence under the control of such apromoter, one positions the 5′ end of the transcription initiation siteof the transcriptional reading frame of the protein between about 1 andabout 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.Also, Saccharomyces cevevisiae yeast expression vector systems, such aspYES2, will also produce HAS under control of the GAL promoter as shownin FIG. 7. FIG. 7 shows that the spHAS enzyme was produced inrecombinant yeast using the pYES2 plasmid. When supplied with UDP-GlcAand UDP-GlcNAc, the enzyme makes high molecular weight HA.

[0148] Where eukaryotic expression is contemplated, one will alsotypically desire to incorporate into the transcriptional unit whichincludes the HAS gene or DNA, an appropriate polyadenylation site (e.g.,5′-AATAAA-3′) if one was not contained within the original clonedsegment. Typically, the poly A addition site is placed about 30 to 2000nucleotides “downstream” of the termination site of the protein at aposition prior to transcription termination.

[0149] It is contemplated that virtually any of the commonly employedhost cells can be used in connection with the expression of HAS inaccordance herewith. Examples of preferred cell lines for expressing HAScDNA of the present invention include cell lines typically employed foreukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI38, BHK, COS-7, RIN and MDCK cell lines. This will generally include thesteps of providing a recombinant host bearing the recombinant DNAsegment encoding the HAS enzyme and capable of expressing the enzyme;culturing the recombinant host in media under conditions that will allowfor transcription of the cloned HAS gene or cDNA and appropriate for theproduction of the hyaluronic acid; and separating and purifying the HASenzyme or the secreted hyaluronic acid from the recombinant host.

[0150] Generally, the conditions appropriate for expression of thecloned HAS gene or cDNA will depend upon the promoter, the vector, andthe host system that is employed. For example, where one employs the lacpromoter, one will desire to induce transcription through the inclusionof a material that will stimulate lac transcription, such asisopropylthiogalactoside. For example, the cloned seHAS gene of thepresent invention is expressed as a HIS₆ containing protein in E. colias shown in FIG. 5. Where other promoters are employed, differentmaterials may be needed to induce or otherwise up-regulatetranscription.

[0151]FIG. 5 depicts the overexpression of recombinant seHAS and spHASin E. coli. Membrane proteins (5 mg per lane) were fractionated bySDS-PAGE using a 10% (w/v) gel under reducing conditions. The gel wasstained with Coomassie blue R-250, photographed, scanned, andquantitated using a molecular dynamics personal densitometer (model PDSIP60). The position of HA synthase is marked by the arrow. Lane A isnative spHAS (Group A); Lane C is native seHAS; Lane E is recombinantseHAS; Lane P is recombinant spHAS; Lane V is vector alone. Standardsused were Bio-rad low Mr and shown in kDa.

[0152] In addition to obtaining expression of the synthase, one willpreferably desire to provide an environment that is conducive to HAsynthesis by including appropriate genes encoding enzymes needed for thebiosynthesis of sugar nucleotide precursors, or by using growth mediacontaining substrates for the precursor-supplying enzymes, such asN-acetylglucosamine or glucosamine (GlcNAc or GlcNH₂) and glucose (Glc).

[0153] One may further desire to incorporate the gene in a host which isdefective in the enzyme hyaluronidase, so that the product synthesizedby the enzyme will not be degraded in the medium. Furthermore, a hostwould be chosen to optimize production of HA. For example, a suitablehost would be one that produced large quantities of the sugar nucleotideprecursors to support the HAS enzyme and allow it to produce largequantities of HA. Such a host may be found naturally or may be made by avariety of techniques including mutagenesis or recombinant DNAtechnology. The genes for the sugar nucleotide synthesizing enzymes,particularly the UDP-Glc dehydrogenase required to produce UDP-GlcA,could also be isolated and incorporated in a vector along with the HASgene or cDNA. A preferred embodiment of the present invention is a hostcontaining these ancillary recombinant gene or cDNAs and theamplification of these gene products thereby allowing for increasedproduction of HA.

[0154] The means employed for culturing of the host cell is not believedto be particularly crucial. For useful details, one may wish to refer tothe disclosure of U.S. Pat. Nos. 4,517,295; 4,801,539; 4,784,990; or4,780,414; all incorporated herein by reference. Where a prokaryotichost is employed, such as S. equisimilis, one may desire to employ afermentation of the bacteria under anaerobic conditions in CO₂-enrichedbroth growth media. This allows for a greater production of HA thanunder aerobic conditions. Another consideration is that Streptococcalcells grown anaerobically do not produce pyrogenic exotoxins.Appropriate growth conditions can be customized for other prokaryotichosts, as will be known to those of skill in the art, in light of thepresent disclosure.

[0155] Once the appropriate host has been constructed, and culturedunder conditions appropriate for the production of HA, one will desireto separate the HA so produced. Typically, the HA will be secreted orotherwise shed by the recombinant organism into the surrounding media,allowing the ready isolation of HA from the media by known techniques.For example, HA can be separated from the cells and debris by filteringand in combination with separation from the media by precipitation byalcohols such as ethanol. Other precipitation agents include organicsolvents such as acetone or quaternary organic ammonium salts such ascetyl pyridinium chloride (CPC).

[0156] A preferred technique for isolation of HA is described in U.S.Pat. No. 4,517,295, and which is incorporated herein by reference, inwhich the organic carboxylic acid, trichloroacetic acid, is added to thebacterial suspension at the end of the fermentation. The trichloroaceticacid causes the bacterial cells to clump and die and facilitates theease of separating these cells and associated debris from HA, thedesired product. The clarified supernatant is concentrated and dialyzedto remove low molecular weight contaminants including the organic acid.The aforementioned procedure utilizes filtration through filtercassettes containing 0.22 μm pore size filters. Diafiltration iscontinued until the conductivity of the solution decreases toapproximately 0.5 mega-ohms.

[0157] The concentrated HA is precipitated by adding an excess ofreagent grade ethanol or other organic solvent and the precipitated HAis then dried by washing with ethanol and vacuum dried, lyophilized toremove alcohol. The HA can then be redissolved in a borate buffer, pH 8,and precipitated with CPC or certain other organic ammonium salts suchas CETAB, a mixed trimethyl ammonium bromide solution at 4 degree(s)Celsius. The precipitated HA is recovered by coarse filtration,resuspended in 1 M NaCl, diafiltered and concentrated as furtherdescribed in the above referenced patent. The resultant HA is filtersterilized and ready to be converted to an appropriate salt, dry powderor sterile solution, depending on the desired end use.

A. Typical Genetic Engineering Methods Which May Be Employed

[0158] If cells without formidable cell membrane barriers are used ashost cells, transfection is carried out by the calcium phosphateprecipitation method, well known to those of skill in the art. However,other methods may also be used for introducing DNA into cells such as bynuclear injection, cationic lipids, electroporation, protoplast fusionor by the Biolistic(tm) Bioparticle delivery system developed by DuPont(1989). The advantage of using the DuPont system is a hightransformation efficiency. If prokaryotic cells or cells which containsubstantial cell wall constructions are used, the preferred method oftransfection is calcium treatment using calcium chloride to inducecompetence or electroporation.

[0159] Construction of suitable vectors containing the desired codingand control sequences employ standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to construct the plasmids required. Cleavage is performedby treating with restriction enzyme (or enzymes) in suitable buffer. Ingeneral, about 1 μg plasmid or DNA fragments are used with about 1 unitof enzyme in about 20 μl of buffer solution. Appropriate buffers andsubstrate amounts for particular restriction enzymes are specified bythe manufacturer. Incubation times of about 1 hour at 370° C. areworkable.

[0160] After incubations, protein is removed by extraction with phenoland chloroform, and the nucleic acid is recovered from the aqueousfraction by precipitation with ethanol. If blunt ends are required, thepreparation is treated for 15 minutes at 15° C. with 10 units ofPolymerase I (Klenow), phenol-chloroform extracted, and ethanolprecipitated. For ligation approximately equimolar amounts of thedesired components, suitably end tailored to provide correct matchingare treated with about 10 units T4 DNA ligase per 0.5 μg DNA. Whencleaved vectors are used as components, it may be useful to preventreligation of the cleaved vector by pretreatment with bacterial alkalinephosphatase.

[0161] For analysis to confirm functional sequences in plasmidsconstructed, the first step was to amplify the plasmid DNA by cloninginto specifically competent E. coli SURE cells (Stratagene) by doingtransformation at 30-32° C. Second, the recombinant plasmid is used totransform E. coli K5 strain Bi8337-41, which can produce the UDP-GlcAprecursor, and successful transformants selected by antibioticresistance as appropriate. Plasmids from the library of transformantsare then screened for bacterial colonies that exhibit HA production.These colonies are picked, amplified and the plasmids purified andanalyzed by restriction mapping. The plasmids showing indications of afunctional HAS gene are then further characterized by any number ofsequence analysis techniques which are known by those of ordinary skillin the art.

B. Source and Host Cell Cultures and Vectors

[0162] In general, prokaryotes were used for the initial cloning of DNAsequences and construction of the vectors useful in the invention. It isbelieved that a suitable source may be Gram-positive cells, particularlythose derived from the Group C Streptococcal strains. Bacteria with asingle membrane, but a thick cell wall such as Staphylococci andStreptococci are Gram-positive. Gram-negative bacteria such as E. colicontain two discrete membranes rather than one surrounding the cell.Gram-negative organisms tend to have thinner cell walls. The singlemembrane of the Gram-positive organisms is analogous to the inner plasmamembrane of Gram-negative bacteria. The preferred host cells areStreptococcus strains that are mutated to become hyaluronidase negativeor otherwise inhibited (EP144019, EP266578, EP244757). Streptococcusstrains that have been particularly useful include S. equisimilis and S.zooepidemicus.

[0163] Prokaryotes may also be used for expression. For the expressionof HA synthase in a form most likely to accommodate high molecularweight HA synthesis, one may desire to employ Streptococcus species suchas S. equisimilis or S. zooepidemicus. The aforementioned strains, aswell as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325),bacilli such as Bacillus subtilis, or other enterobacteriaceae such asSerratia marcescens, could be utilized to generate a “super” HAScontaining host.

[0164] In general, plasmid vectors containing origins of replication andcontrol sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries an origin of replication, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using pBR322, a plasmidderived from an E. coli species. pBR322 contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. A pBR plasmid or a pUC plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, promoterswhich can be used by the microbial organism for expression of its ownproteins.

[0165] Those promoters most commonly used in recombinant DNAconstruction include the lacZ promoter, tac promoter, the T7bacteriophage promoter, and tryptophan (trp) promoter system. Whilethese are the most commonly used, other microbial promoters have beendiscovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors. Also for use with the presentinvention one may utilize integration vectors.

[0166] In addition to prokaryotes, eukaryotic microbes, such as yeastcultures may also be used. Saccharomyces cerevisiae, or common baker'syeast is the most commonly used among eukaryotic microorganisms,although a number of other strains are commonly available. Forexpression in Saccharomyces, the plasmid YRp7, for example, is commonlyused. This plasmid already contains the trp1 gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow without tryptophan, for example, ATCC No. 44076 or PEP4-1. Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Suitablepromoting sequences in yeast vectors include the promoters for thegalactose utilization genes, the 3-phosphoglycerate kinase or otherglycolytic enzymes, such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0167] In constructing suitable expression plasmids, the terminationsequences associated with these genes are also ligated into theexpression vector 3′ of the sequence desired to be expressed to providepolyadenylation of the mRNA and termination. Other promoters, which havethe additional advantage of transcription controlled by growthconditions are the promoter region for alcohol dehydrogenase 2,cytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization. Any plasmid vector containing a yeast-compatible promoter,origin of replication and termination sequences is suitable.

[0168] In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture has become a routineprocedure in recent years. Examples of such useful host cell lines areVERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI38,BHK, COS, and MDCK cell lines.

[0169] For use in mammalian cells, the control functions on theexpression vectors are often provided by viral material. For example,commonly used promoters are derived from polyoma, Adenovirus 2, bovinepapilloma virus and most frequently Simian Virus 40 (SV40). The earlyand late promoters of SV40 virus are particularly useful because bothare obtained easily from the virus as a fragment which also contains theSV40 viral origin of replication. Smaller or larger SV40 fragments mayalso be used, provided there is included the approximately 250 bpsequence extending from the Hind III site toward the Bg1 I site locatedin the viral origin of replication.

[0170] Further, it is also possible, and often desirable, to utilizepromoter or control sequences normally associated with the desired genesequence, provided such control sequences are compatible with the hostcell systems. An origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, BPV) source,or may be provided by the host cell chromosomal replication mechanism.If the vector is integrated into the host cell chromosome, the lattermechanism is often sufficient.

C. Isolation of a bona fide HA synthase gene from a highly encapsulatedstrain of Group C Streptococcus equisimilis

[0171] The encoded protein, designated seHAS, is 417 amino acids(calculated molecular weight of 47,778 and pI of 9.1) and is thesmallest member of the HAS family identified thus far (FIG. 2). seHASalso migrates anomalously fast in SDS-PAGE (M_(r)˜42 kDa) (FIGS. 5 and8).

[0172]FIG. 8 is a graphical representation of a Western Blot analysis ofrecombinant seHAS using specific antibodies. Group C (C; lane 1) orGroup A (A; lane 4) Streptococcal membranes and E. coli membranes (9mg/lane) containing recombinant seHAS (E; lanes 2, 7, and 9) or spHAS(P; lanes 3, 6, 8, and 10) were fractionated by reducing SDS-PAGE andelectrotransferred to nitrocellulose. Strips of nitrocellulose wereprobed and developed as described in the application using purified IgGfractions raised to the following regions of spHAS: central domainpeptide E¹⁴⁷-T¹⁶¹ (lanes 1-4); C-terminus peptide (lanes 5-6); thecomplete protein (lanes 7 and 8); recombinant central domain (lanes 9and 10). Nonimmune IgG or membranes from cells transformed with vectoralone gave no staining as in lane 5.

[0173] The seHAS and spHAS protein (previously identified in U.S. Ser.No. 08/899,940) encoding sequences are 72% identical. The deducedprotein sequence of seHAS was confirmed by reactivity with a syntheticpeptide antibody (FIG. 8). Recombinant seHAS expressed in E. coli wasrecovered in membranes as a major protein (FIG. 5) and synthesized verylarge molecular weight HA in the presence of UDP-GlcNAc and UDP-GlcA invitro (FIG. 9).

[0174]FIG. 9 shows a kinetic analysis of the HA size distributionsproduced by seHAS and spHAS. E. coli membranes containing equal amountsof seHAS or spHAS protein were incubated at 37° C. with 1.35 mMUDP-[¹⁴C] GlcA (1.3×10³ dpm/nmol) and 3.0 mM UDP-GlcNAc as described inthe application. These substrate concentrations are greater than 15times the respective Km valves. Samples taken at 0.5, 1.0, and 60 minwere treated with SDS and chromatographed over Sephacryl S400 HR. The HAprofiles in the fractionation range of the column (fractions 12-24) arenormalized to the percent of total HA in each fraction. The values abovethe arrows in the top panel are the MWs (in millions) of HA determineddirectly in a separate experiment using a Dawn multiangle laser lightscattering instrument (Wyatt Technology Corp.). The size distributionsof HA synthesized by seHAS (,▪,▴) and spHAS (◯, □,_) at 0.5 min (◯,),1.0 min (□,▪) and 60 min (_,▴) are shown as indicated. Analysis showedthat seHAS and spHAS are essentially identical in the size distributionof HA chains they synthesize (FIG. 9). SeHAS is twice as fast as spHASin its ability to make HA.

C.1 Bacterial strains and vectors

[0175] The mucoid group C strain D181; (Streptococcus equisimilis) wasobtained from the Rockfeller University Collection. The E. coli hoststrains Sure and XL1-Blue MRF′ were from Stratagene and strain Top10 F′was from Invitrogen. Unless otherwise noted, Streptococci were grown inTHY and E. coli strains were grown in LB medium. pKK-223 Expressionvector was from Pharmacia, PCR 2.1 cloning vector was from Invitrogen,and predigested λ Zap Express TM Bam HI/CIAP Vector was from Stratagene.

Recombinant DNA and Cloning

[0176] High molecular mass Genomic DNA from Streptococcus equisimilisisolated by the method of Caparon and Scott (as known by those withordinary skill in the art) was partially digested with Sau3Al to anaverage size of 2-12 kb. The digested DNA was precipitated with ethanol,washed and ligated to the Bam HI/CIAPX Zap Express vector. Ligated DNAwas packaged into phage with a Packagene™ extract obtained from Promega.The titer of the packaged phage library was checked using XL1-Blue MRF′E. coli as a host.

C.3 Degenerate PCR Amplification

[0177] Degenerate oligonucleotides were designed based upon conservedsequences among spHAS (Streptococcus pyogenes), DG42 (Xenopus laevisHAS; 19) and nodC (a Rhizobium meliloti modulation factor; 20) and wereused for PCR amplification with D181 genomic DNA as a template.Amplification conditions were 34 cycles at: 94° C. for 1 min, 44° C. for1 min, 72° C. for 1.5 min followed by a final extension at 72° C. for 10min. Oligonucleotide HADRF1. 5′-GAY MGA YRT YTX ACX AAT TAY GCT ATH GAYTTR GG-3′ (sense strand) corresponds to the sequence D²⁵⁹RCLTNYAIDL(spHAS). Oligonucleotide HACTR1, 5′-ACG WGT WCC CCA NTC XGY ATT TTT NADXGT RCA-3′ (antisense strand) corresponds to the region C⁴⁰⁴TIKNTEWGTR(spHAS). The degeneracy of bases at some positions are represented bynomenclature adopted by the IUPAC in its codes for degenerate baseslisted in Table IV. TABLE IV IUPAC Codes - Degenerate Bases TheInternational Union for Pure and Applied Chemistry (IUPAC) hasestablished a standard single-letter designation for degenerate bases.These are: B = C + G + T D = A + G + T H = A + C + T K = T + G M = A + CN = A + C + G + T R = A + G S = G + C W = A + T V = A + C + G X = aminor bases (specified elsewhere) Y = C + T

[0178] These two oligonucleotides gave a 459 bp PCR product, which wasseparated on an agarose gel and purified using the BIO-101 Genecleankit. This fragment was then cloned into PCR2.1 vector using TOP 10 F′cells as a host according to the manufacturer's directions. Doublestranded plasmid DNA was purified from E. coli (Top 10 F′) using theQIAfilter Plasmid Midi Kit (Qiagen). Two other degenerate sense primerswere also synthesized: HAVAF1, 5′-GTN GCT GCT GTW RTX CCW WSX TWT AAYGAR GA-3′ (corresponding to the region V⁶⁶AAVIPSYNE of spHAS) andHAVDF1, 5′-GTX RWT GAY GGN WSX WSN RAX GAT GAX GC-3′ (based onV¹⁰⁰DDGSSNTD of spHAS). Two unique antisense primers were synthesizedbased on the sequence of the 459 bp PCR product. These were: D181.2,5′-GAA GGA CTT GTT CCA GCG GT-3′ and D181.4, 5′-TGA ATG TTC CGA CAC AGGGC-3′, Each of the two degenerate sense primers, when used with eitherD181.2 or D181.4 to amplify D181 genomic DNA, gave expected size PCRproducts. The four PCR products were cloned and sequenced using the samestrategy as above. For each PCR product, sequences obtained from sixdifferent clones were compared in order to derive a consensus sequence.Thus we obtained a 1042 bp sequence with a continuous ORF with highhomology to spHAS.

C.4 Library Screening

[0179] molecular probes were used to screen the library; the cloned 459bp PCR product and oligonucleotide D181.5(5′-GCTTGATAGGTCACCAGTGTCACG-3′; derived from the 1042 bp sequence). The459 bp PCR product was radiolabeled using the Prime-It 11 random primerlabeling Kit (Stratagene) according to the manufacturers instructions.Oligonucleotides were labeled by Kinace-It Kinasing Kit (Stratagene)using [γ³² P]ATP. Radiolabeled products were separated from nonlabeledmaterial on NucTrap Push columns (Stratagene). The oligoprobe hybridizedspecifically with a D181 genomic digest on Southern blots. To screen theλ phage library, XLBLUE MRF′ was used as a host (3000 plaques/plate) onNitrocellulose membranes containing adsorbed phage, were prehybridizedat 60° C. and hybridized with 5′-end labeled oligonucleotide, D181.5, inQuikHyb Hybridization solution (Stratagene) at 80° C. according toinstructions.

[0180] The membranes were then washed with 2×SSC buffer and 0.1% (w/v)SDS at room temperature for 15 min, at 60° C. with 0.1×SSC buffer and0.1% SDS (w/v) for 30 min, dried and then exposed to Bio-Max MS filmovernight at −70° C. Positive plaques were replated and rescreenedtwice. Pure positive phages were saved in SM buffer with chloroform. PCRon these phages with vector primers revealed 3 different insert sizes.

[0181] PCR with a combination of vector primers and primers fromdifferent regions of the cloned 1042 bp sequence revealed that only oneof the three different phages had the complete HAS gene. The insert sizein this phage was 6.5 kb. Attempts to subclone the insert into plasmidform by autoexcision from the selected phage library clone failed.Therefore, a PCR strategy was applied again on the pure positive phageDNA to obtain the 5′ and 3′ end of the ORF. Oligonucleotide primersD181.3 (5′-GCCCTGTGTCGGAACATTCA-3′) and T3 (vector primer) amplified a3kb product and oligonucleotides D181.5 and T7 (vector primer) amplifieda 2.5 kb product. The 5′ and 3′-end sequences of the ORF were obtainedby sequencing these two above products. Analysis of all PCR productsequences allowed us to reconstruct the ORF of the 1254 bp seHAS gene.

C.5 Expression cloning of the seHAS

[0182] Primers were designed at the start and stop codon regions ofseHAS to contain an EcoR1 restriction site in the sense oligonucleotide(5′-AGGATCCGAATTCATGAGAACATTAAAAAACCTC-3′) and a Pst1 site in theantisense oligonucleotide (5′-AGAATTCTGCAGTTATAATAATTTTTTACGTGT-3′).These primers amplified a 1.2 kb PCR product from D181 genomic DNA aswell as from pure hybridization-positive phage. The 1.2 kb product waspurified by agarose gel electrophoresis, digested with Pst1 and EcoR1and cloned directionally into Pst1-and EcoR1-digested pKK223 vector. Theligated vector was transformed into E. coli SURE cells that were thengrown at 30° C. This step was practically important since other hostcells or higher temperatures resulted in deletions of the cloned insert.Colonies were isolated and their pDNA purified. Out of six colonies(named a,b,c,d,e, and f), five had the correct size insert, while onehad no insert.

C.6 HA Synthase Activity

[0183] HA synthase activity was assayed in membranes prepared from the 5above clones. Fresh log phase cells were harvested at 3000g, washed at4° C. with PBS and membranes were isolated by a modification of aprotoplast method as known by those of ordinary skill in the art.Membrane preparations from Streptococcus pyogenes and Streptococcusequisimilis were also obtained by modification of a different protoplastprocedure. Membranes were incubated at 37° C. in 50 mM sodium andpotassium phosphate, pH 7.0 with 20 mM MgCl₂, 1 mM DTE, 120 μM UDP-GlcAand 300 μM UDP-GlcNAc. Incorporation of sugar was monitored by usingUDP- [¹⁴C] GlcA (318 mCi/mmol; ICN) and/or UDP-[³H] GlcNAc (29.2 Ci/mmolNEN). Reactions were terminated by addition of SDS to a finalconcentration of 2% (w/v). Product HA was separated from precursors bydescending paper chromatography and measured by determining incorporatedradioactivity at the origin.

C.7 Gel Filtration Analysis

[0184] Radiolabeled HA produced in vitro by membranes containingrecombinant seHAS or spHAS was analyzed by chromatography on a column(0.9×40 cm) of Sephacryl S500 HR (Pharmacia Biotech Inc.). Samples (0.4ml in 200 mM NaCl, 5mM Tris-HCl, pH 8.0, plus 0.5% SDS) were eluted with200 mM, NaCl, 5 mM Tris-HCL, and pH 8.0 and 0.5 ml fractions wereassessed for ¹⁴C and/or ³H radioactivity. Authenticity of the HApolysaccharide was assessed by treatment of a separate identical samplewith the HA-specific hyaluronate lyase of Streptomyces hyalurolyticus(EC 4.2.2.1) at 370° C. for 3 hrs. The digest was then subjected to gelfiltration.

C.8 SDS-PAGE and Western Blotting

[0185] SDS-PAGE was performed according to the Laemmli method.Electrotransfers to nitrocellulose were performed within standardblotting buffer with 20% methanol using a Bio-Rad mini Transblot device.The blots were blocked with 2% BSA in TBS. Protein A/G alkalinephosphatase conjugate (Pierce) and p-nitrobluetetrazolium/5-bromo-4-chloro-3 indolyl phosphate p-toluidine salt wereused for detection.

C.9 DNA Sequence and Analysis

[0186] Plasmids were sequenced on both strands using fluorescent labeledvector primers. Sequencing reactions were performed using aThermosequenase™ kit for fluorescent labeled primers (with 7-deazaG).Samples were electrophoresed on a Pharmacia ALF Express DNA Sequencerand data were analyzed by the ALF Manager Software v3.02. Internalregions of inserts were sequenced with internal primers using the ABIPrism 377 (Software version 2.1.1). Ambiguous regions were sequencedmanually using Sequenase™ 7-deaza—DNA polymerase, 7-deaza GTP master mix(USB) and [α-³⁵S] DATP (Amersham Life Sciences). The sequences obtainedwere compiled and analyzed using DNASIS, v2.1 (Hitachi SoftwareEngineering Co., Ltd.). The nucleotide and amino acid sequences werecompared with other sequences in the Genbank and other databases.

C.10 Identification of seHAS

[0187] Identification of seHAS was accomplished by utilizing a PCRapproach with oligonucleotide primers based on several regions of highidentity among spHAS, DG42 (now known to be a developmentally regulatedX. laevis HAS and designated xlHAS) and NodC (a Rhizobium β-GlcNActransferase). The xlHAS and NodC proteins are, respectively, ˜50% and˜10% identical to spHAS. This strategy yielded a 459 bp PCR productwhose sequence was 66.4% identical to spHAS, indicating that a Group Chomologue (seHAS) of the Group A (spHAS) HA synthase gene had beenidentified. The complete coding region of the gene was thenreconstructed using a similar PCR-based strategy. A final set of PCRprimers was then used to amplify the complete ORF from genomic DNA. Whenthis 1.2 kb PCR fragment was incorporated into the expression vectorpKK223 and transformed into E. coli SURE cells, HA synthetic activitywas demonstrated in isolated membranes from 5 of the 5 colonies tested.

[0188] The ORF of the reconstructed gene encodes a novel predictedprotein of 417 amino acids that was not in the database and it is twoamino acids shorter than spHAS. The two bacterial proteins are 72%identical and the nucleic acid sequences are 70% identical. Thepredicted molecular weight of the seHAS protein is 47,778 and thepredicted isoelectric point is at pH 9.1. Three recently identifiedmammalian HASs (muHAS1, muHAS2, muHAS3, FIG. 2) are similar to thebacterial proteins. The overall identity between the two groups is˜28-31%, and in addition many amino acids in seHAS are highly conservedwith those of the eukaryotic HASs (e.g. K/R or D/E substitutions). A98R,the PBCY-1 HAS is 28-33 percent identical to the mammalian HASS, and ispredicted to have a similar topology in the lipid membrane. Withinmammalian species the same family members are almost completelyidentical (e.g. muHAS1 and huHAS1 are 95% identical; muHAS2 and huHAS2are 98% identical). However, and as shown in FIG. 3, even within thesame species the different HAS family members are more divergent (e.g.muHAS1 and muHAS2 are 53% identical; muHAS1 and muHAS3 are 57%identical; muHAS2 and muHAS3 are 71% identical).

[0189]FIG. 10 shows hydropathy plots for seHAS and predicted membranetopology. The hydrophilicity plot for the Streptococcal Group C HAS wasgenerated by the method of Kyte and Doolittle (J. Mol. Biol. 157, 105,1982) using DNAsIs. The protein is predicted to be an integral membraneprotein.

[0190]FIG. 11 shows a model for the topologic organization of seHAS inthe membrane. The proposed topology for the protein conforms to thecharge-in rule and puts the large central domain inside. This domain islikely to contain most of the substrate binding and catalytic functionsof the enzyme. Cys²²⁶ in seHAS, which is conserved in all HAS familymembers, as well as the other three cysteines are shown in the centraldomain. CyS²⁸¹ is a critical residue whose alteration can dramaticallyalter the size distribution of HA product synthesized by the enzyme.

[0191] The overall membrane topology predicted for seHAS is identical tothat for spHAS and the eukaryotic HASs reported thus far. The proteinhas two putative transmembrane domains at the amino terminus and 2-3membrane-associated or transmembrane domains at the carboxyl end. Thehydropathy plots for the two Streptococcal enzymes are virtuallyidentical and illustrate the difficulty in predicting the topology ofthe extremely hydrophobic region of -90 residues at K³¹³ R⁴⁰⁶ in seHAS(K³³³ -K⁴⁰⁵ in spHAS).

[0192] seHAS was efficiently expressed in E. coli cells. Roughly 10% ofthe total membrane protein was seHAS as assessed by staining of SDS-PAGEgels (FIG. 5). The prominent seHAS band at 42 kD is quantitativelymissing in the vector-only control lane. This unusually high level ofexpression for a membrane protein is also found for spHAS, using thesame vector in SURE cells. About 8% of the membrane protein is spHAS inE. coli SURE cells. In contrast, the amount of seHAS in Group Cmembranes is not more than 1% of the total membrane protein. The spHASin Group A membranes is barely detectable. The recombinant seHASexpressed in E. coli SURE cells does not synthesize HA in vivo, sincethese cells lack UDP-GlcA, one of the required substrates. Membranes,however containing the recombinant seHAS protein synthesize HA whenprovided with the substrates UDP-GlcNAc and UDP-GlcA (FIG. 12).

[0193]FIG. 12 shows the synthesis of authentic HA by recombinant seHAS.E. coli membranes (69 μg) prepare from cells containing recombinantseHAS or vector alone were incubated at 37° C. for 1 hour with 700 μMUDP-[³H]GlcNAc (2.78×10³ dpm/nmol; □,▪) and 300 μM UDP-[¹⁴C]GlcA(3.83×10³ dpm/nmol; ◯,) in a final volume of 200 μl as describedherein. The enzyme reaction was stopped by addition of EDTA to a finalconcentration of 25 mM. Half the reaction mix was treated withStreptomyces hyaluronidase at 37° C. for 3 hours. SDS (2%, w/v) wasadded to hyaluronidase-treated (◯,□) and untreated (,▪) samples, whichwere heated at 90° C. for 1 min. The samples were diluted to 500 μl withcolumn buffer (5 mM Tris, 0.2 M Nacl, pH 8.0), clarified bycentrifugation and 200 μl was injected onto a Sephacryl S-500 HR column.Fractions (1 ml) were collected and radioactivity was determined. BD isthe peak elution position position of blue dextran (˜2×10⁶ DA;Pharmacia). V_(o) marks the excluded volume and V_(i) the includedvolume. The ratio of [¹⁴c] GlcA: [³H] GlcNAc incorporated into the totalamount of HA fractionated on the column is 1.4, which is identical tothe ratio of specific activities of the two substrates. Therefore, themolar ratios of the sugars incorporated into product is 1:1 as predictedfor authentic HA. Membranes from cells transformed with vector alone didnot synthesize HA.

[0194] Using 120 μM UDP-GlcA and 300 μM UDP-GlcNAc, HA synthesis waslinear with membrane protein (at ≦0.2 μg) and for at least 1 hour. Also,membranes prepared from nontransformed cells or cells transformed withvector alone have no detectable HAS activity. HA synthesis is negligibleif Mg⁺² is chelated with EDTA (<5% of control) or if either of the twosubstrates are omitted (˜2% of control). Recombinant seHAS also showedthe expected specificity for sugar nucleotide substrates, being unableto copolymerize either UDP-GalA, UDP-Glc or UDP-GalNAc with either ofthe two normal substrates (Table II).

[0195] Based on gel filtration analysis, the average mass of the HAsynthesized by seHAS in isolated membranes is 5-10×10⁶ Da. The productof the recombinant seHAS is judged to be authentic HA based on theequimolar incorporation of both sugars and its sensitivity todegradation by the specific Streptomyces hyaluronidase (FIG. 12).Although the conditions for total HA synthesis were not optimal (since˜90% of one substrate was incorporated into product), the enzymeproduced a broad distribution of HA chain lengths. The peak fractioncorresponds to an HA mass of 7.5×10⁶ Da which is a polymer containingapproximately 36,000 monomeric sugars. The distribution of HA sizesresolved on this column ranged from 2-20×10⁶ Da.

[0196] The deduced protein sequence of seHAS was confirmed by theability of antibodies to the spHAS protein to cross-react with the GroupC protein (FIG. 8). Polyclonal antibodies to the whole spHAS protein orto just the central domain of spHAS also reacted with the seHAS protein.Antipeptide antibody to the C-terminus of spHAS did not cross-react withthis somewhat divergent region in the seHAS protein. However,antipeptide antibody directed against the spHAS sequence E¹⁴⁷-T¹⁶¹recognized the same predicted sequence in seHAS. The antipeptideantibody also reacts with the native seHAS and spHAS proteins inStreptococcal membranes and confirms that the native and recombinantenzymes from both species are of identical size. Like the spHAS protein,seHAS migrates anomalously fast on SDS-PAGE. Although the calculatedmass is 47,778 Da, the M_(r) by SDS-PAGE is consistently ˜42 kDa.

[0197] Because of the sequence identity within their central domainregions and the overall identical structure predicted for the twobacterial enzymes, the peptide-specific antibody against the regionE¹⁴⁷-T¹⁶¹ can be used to normalize for HAS protein expression inmembranes prepared from cells transformed with genes for the twodifferent enzymes. Using this approach, membranes with essentiallyidentical amounts of recombinant spHAS or seHAS were compared withrespect to the initial rate of HA synthesis and the distribution of HAproduct size.

[0198] As shown for spHAS, the synthesis of HA chains by seHAS isprocessive. The enzymes appear to stay associated with a growing HAchain until it is released as a final product. Therefore, it is possibleto compare the rates of HA elongation by seHAS and spHAS by monitoringthe size distribution of HA chains produced at early times, during thefirst round of HA chain synthesis. Based on gel filtration analysis ofHA product sizes at various times, we estimated that the average rateelongation by seHAS is about 9,000 monosaccharides/minute at 37° C.(FIG. 9). In five minutes, the enzymes can polymerize an HA chain of5-10×10⁶ Da. During a 60 min incubation, therefore, each enzyme moleculecould potentially initiate, complete and release on the order of 5-8such large HA molecules. At early times (e.g. ≦1 min), reflectingelongation of the first HA chains, the size distribution of HA producedby seHAS was shifted to larger species compared to spHAS. By 60 min thetwo distributions of HA product sizes are indistinguishable.

[0199] The cloned seHAS represents the authentic Group C HA synthase.Previously reported or disclosed “Group C” proteins are, therefore, notthe true Group C HAS. The seHAS protein is homologous to nine of thecurrently known HA synthases from bacteria, vertebrates, and a virusthat now comprise this rapidly growing HA synthase family. This homologyis shown particularly in FIG. 2. In mammals three genes, designated HAS1, HAS 2 and HAS 3, have been identified and mapped to three differentchromosomes in both human and mouse. In amphibians the only HAS proteinidentified thus far is the developmentally regulated DG42, which wascloned in 1988 and recently shown to encode the HA synthase activity byanalysis of the recombinant protein in yeast membranes. Probably otherX. laevus HAS genes will soon be identified.

[0200] A divergent evolution model suggests that a primitive bacterialHAS precursor may have been usurped early during vertebrate developmentor the bacterial pathogenic strategy of making an HA capsule wasdeveloped when a primitive bacteria captured in primordial HAS.Convergent evolution of the bacterial and eukaryotic HAS enzymes to acommon structural solution seems unlikely, but may have occurred.

[0201] None of the three mammalian isozymes for HAS have yet beencharacterized enzymatically with respect to their HA product size. Atleast ten identified HAS proteins are predicted to be membrane proteinswith a similar topology. HA synthesis occurs at the plasma membrane andthe HA is either shed into the medium or remains cell associated to formthe bacterial capsule or a eukaryotic pericellular coat. The sugarnucleotide substrates in the cytoplasm are utilized to assemble HAchains that are extruded through the membrane to the external space.

[0202] The protein topology in the very hydrophobic carboxyl portion ofthe HAS protein appears to be critical in understanding how the enzymesextend the growing HA chain as it is simultaneously extruded through themembrane. For example, the unprecedented enzymatic activity may requireunusual and complex interactions of the protein with the lipid bilayer.Preliminary results based on analysis of spHAS-alkaline phosphatasefusion proteins indicate that the amino and carboxyl termini and thelarge central domains are all intracellular, as shown in FIGS. 10 and11. The seHAS protein also contains a large central domain (˜63% of thetotal protein) that appears to contain the two substrate binding sitesand the two glycosyltransferase activities needed for HA synthesis.Although current software programs cannot reliably predict the number ornature of membrane-associated domains within the long C-terminalhydrophobic stretch, the proposed topological arrangement agrees withthe present evidence and applies as well to the eukaryotic enzymes,which are -40% larger primarily due to extention of the C-terminal endof the protein with 2 additional predicted transmembrane domains.

[0203] Four of the six Cys residues in spHAS are conserved with seHAs.Only Cys225 in both bacterial enzymes is conserved in all members of theHAS family. Since sulfhydryl reactive agents, such as p-mercurobenzoateor NEM, greatly inhibit HAS activity, it is likely that this conservedCys is necessary or important for enzyme activity. Initial results fromsite-directed mutagenesis studies, however, indicate that a C225S mutantof spHAS is not inactive, it retains 5-10% of wildtype activity.

[0204] The recognition of nucleic acid sequences encoding only seHAS,only spHAS, or both seHAS and spHAS using specific oligonucleotides isshown in FIG. 13. Three pairs of sense-antisense oligonucleotides weredesigned based on the sequence of ID SEQ NO. 1 and the coding sequencefor spHAS. The seHAS based nucleic acid segments (se1-se2 andsesp1-sesp2) are indicated in FIG. 14. These three oligonucleotide pairswere hybridized under typical PCR reactions with genomic DNA from eitherGroup C (seHAS) (lanes 2, 4, and 6) or Group A (spHAS) (lanes 3,5, and7) streptococci. Lanes 1 and 8 indicate the positions of MW standards inkb (kilobases). The PCR reactions were performed using Taq DNApolymerase (from Promega) for 25 cycles as follows:94 degrees Celsiusfor 1 minute to achieve DNA denaturation, 48 degrees Celsius (42 degreesCelsius for the smaller common sesp primers) for 1 minute to allowhybridization, and 72 degrees Celsius for 1.5 minutes for DNA synthesis.The PCR reaction mixtures were then separated by electrophoresis on a 1%agarose gel.

[0205] The se1-se2 primer pair was designed to be uniquely specific forthe Group C HAS (seHAS). The sp1-sp2 primer pair was designed to beuniquely specific for the Group A HAS (spHAS). The sesp1-sesp2 primerpair was designed to hybridize to both the Group A and Group C HASnucleic acid sequences. All three primer pairs behaved as expected,showing the appropriate ability to cross-hybridize and support thegeneration of PCR products that were specific and/or unique.

[0206] The oligonucleotides used for specific PCR or hybridization areshown in FIG. 14. The synthetic oligonucleotides of SEQ ID NOS:3, 4, 5,and 6 are indicated in the corresponding regions of SEQ ID NO. 1. Theseregions are in bold face and marked, respectively as primers se1, se2,sesp1, and sesp2. The #1 indicates primers in the sense direction, whilethe #2 indicates a primer in the antisense direction. Each of the fouroligonucleotides will hybridize specifically with the seHAS sequence andthe appropriate pairs of sense/antisense primers are suitable for use inthe polymerase chain reaction as shown in FIG. 13.

[0207]FIG. 7 shows a gel filtration analysis of hyaluronic acidsynthesized by recombinant HAS expressed in yeast membranes. A DNAfragment encoding the open reading frame of 419 amino acid residuescorresponding to spHAS (with the original Val codon switched to Met) wassubcloned by standard methods in the pYES2 yeast expression vector (fromInvitrogen) to produce pYES/HA. Membranes from cells with this constructwere prepared by agitation with glass beads. The samples derived frompYES/HA constructs contained substantial HA synthase activity and the“42 kDa” HAS protein was detected by Western analysis using specificantibodies; membranes from cells with vector alone possessed neitheractivity nor the immunoreactive band (not shown). Membranes (315 ugprotein) were first incubated with carrier free UDP-[¹⁴C]GlcA (1 uCi¹⁴C)amd 900 uM unlabeled UDP-GlcNAc in 50 mM Tris, pH 7, 20 mM MgCl2, 1mMDTT, and 0.05 M NaCl (450 ul reaction volume) at 30 degrees Celsius for1.5 minutes. After this pulse-label period nonradiolabeled UDP-GlcA wasthen added to final concentrations of 900 uM. Samples (100 uL) weretaken after the pulse at 1.5 min (dark circle), and 15 (black square),and 45 (black triangle) min after the “chase.” The reactions wereterminated by the addition of SDS to 2% and heating at 95 degreesCelsius for 1 min. The samples were clarified by centrifugation(10,000×g, 5 min) before injection of half of the sample onto aSephacryl S-500HR gel filtration column (Pharmacia; 1×50 cm)equilibrated in 0.2 M NaCl, 5 mM Tris, pH 8.

[0208] The column was eluted at 0.5 ml/min and radioactivity in thefractions (1 ml) was quantitated by liquid scintillation counting afteradding BioSafeII cocktail (4.5 ml, Research Products Intl.). The voidvolume and the totally included volumes were at elution volumes of 14 mland 35.5 ml, respectively. The peak of blue dextran (average 2×10⁶ Da)eluted at 25-27 ml. The recombinant HAS expressed in the eukaryoticyeast cells makes high molecular weight hyaluronic acid in vitro.

[0209] Thus it should be apparent that there has been provided inaccordance with the present invention a purified nucleic acid segmenthaving a coding region encoding enzymatically active HAS, methods ofproducing hyaluronic acid from the seHAS gene, and the use of hyaluronicacid produced from a HAS encoded by the seHAS gene, that fully satisfiesthe objectives and advantages set forth above. Although the inventionhas been described in conjunction with specific embodiments thereof, itis evident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications, and variations that fallwithin the spirit and broad scope of the appended claims.ATGAGAACATTAAAAAACCTCATAACTGTTGTGGCCTTTAGTATTTTTTGGGTACTGTTGATTTACGTCAAT72 SEQUENCE ID NO. 1GTTTATCTCTTTGGTGCTAAAGGAAGCTTGTCAATTTATGGCTTTTTGCTGATAGCTTACCTATTAGTCAAA144ATGTCCTTATCCTTTTTTTACAAGCCATTTAAGGGAAGGGCTGGGCAATATAAGGTTGCAGCCATTATTCCC216TCTTATAACGAAGATGCTGAGTCATTGCTAGAGACCTTAAAAAGTGTTCAGCAGCAAACCTATCCCCTAGCA288GAAATTTATGTTGTTGACGATGGAAGTGCTGATGAGACAGGTATTAAGCGCATTGAAGACTATGTGCGTGAC360ACTGGTGACCTATCAAGCAATGTCATTGTTCATCGGTCAGAGAAAAATCAAGGAAAGCGTCATGCACAGGCC432TGGGCCTTTGAAAGATCAGACGCTGATGTCTTTTTGACCGTTGACTCAGATACTTATATCTACCCTGATGCT504TTAGAGGAGTTGTTAAAAACCTTTAATGACCCAACTGTTTTTGCTGCGACGGGTCACCTTAATGTCAGAAAT576AGACAAACCAATCTCTTAACACGCTTGACAGATATTCGCTATGATAATGCTTTTGGCGTTGAACGAGCTGCC648CAATCCGTTACAGGTAATATCCTTGTTTGCTCAGGTCCGCTTAGCGTTTACAGACGCGAGGTGGTTGTTCCT720AACATAGATAGATACATCAACCAGACCTTCCTGGGTATTCCTGTAAGTATTGGTGATGACAGGTGCTTGACC792AACTATGCAACTGATTTAGGAAAGACTGTTTATCAATCCACTGCTAAATGTATTACAGATGTTCCTGACAAG864ATGTCTACTTACTTGAAGCAGCAAAACCGCTGGAACAAGTCCTTCTTTAGAGAGTCCATTATTTCTGTTAAG936AAAATCATGAACAATCCTTTTGTAGCCCTATGGACCATACTTGAGGTGTCTATGTTTATGATGCTTGTTTAT1008TCTGTGGTGGATTTCTTTGTAGGCAATGTCAGAGAATTTGATTGGCTCAGGGTTTTAGCCTTTCTGGTGATT1080ATCTTCATTGTTGCCCTGTGTCGGAACATTCATTACATGCTTAAGCACCCGCTGTCCTTCTTGTTATCTCCG1152TTTTATGGGGTGCTGCATTTGTTTGTCCTACAGCCCTTGAAATTATATTCTCTTTTTACTATTAGAAATGCT1224 GACTGGGGAACACGTAAAAAATTATTATAA 1254

[0210]

5′- G C T G A T G A G A C A G G T A T T A A G C SEQUENCE ID NO. 3primer: se1 (sense, nucleotides G³¹⁶-C³³⁷)

[0211] 5′-A T C A A A T T C T C T G A C A T T G C SEQUENCE ID NO. 4primer: se2 (antisense, for sense nucleotides G¹⁰³¹-T¹⁰⁵⁰)

[0212] 5′-G A C T C A G A T A C T T A T A T C T A SEQUENCE ID NO. 5primer: sesp1 (sense, for nucleotides G⁴⁷⁵-A⁴⁹⁴)

[0213] 5′-T T T T T A C G T G T T C C C C A SEQUENCE ID NO. 6 primer:sesp2 (antisense, for sense nucleotides T¹²²⁸-A¹²⁴⁴)

[0214] Protein sequence of A98R, the PBCV-1 HA synthase 1 MGKNIIIMVSWYTIITSNLI AVGGASLILA PAITGYVLHW NIALSTIWGV SAYGIFVFGF SEQUENCE ID NO. 761 FLAQVLFSEL NRKRLRKWIS LRPKGWNDVR LAVIIAGYRE DPYMFQKCLE SVRDSDYGNV 121ARLICVIDGD EDDDMRMAAV YKAIYNDNIK KPEFVLCESD DKEGERIDSD FSRDICVLQP 181HRGKRECLYT GFQLAKMDPS VNAVVLIDSD TVLEKDAILE VVYPLACDPE IQAVAGECKI 241WNTDTLLSLL VAWRYYSAFC VERSAQSFFR TVQCVGGPLG AYKDIIKEIK DPWISQRFLG 301QKCTYGDDRR LTNEILMRGK KVVFTPFAVG WSDSPTNVFR YIVQQTRWSK SWCREIWYTL 361FAAWKHGLSG IWLAFECLYQ ITYFFLVIYL FSRLAVEADP RAQTATVIVS TTVALIKCGY 421FSFRAKDIRA FYFVLYTFVY FFCMIPARIT AMMTLWDIGW DTRGGNEKPS VGTRVALWAK 481QYLIAYMWWA AVVGAGVYSI VHNWMFDWNS LSYRFALVGI CSYIVFIVIV LVVYFTGKIT 541TWNFTKLQKE LIEDRVLYDA TTNAQSV                             567

[0215] Nucleotide Sequence of A98R gene in the PBCV-1 Virus GenomeStart: ATG 50901 Stop: TGA 52607 50881 aagacttctt gaaagttaca ATGggtaaaaatataatcat aatggtttcg tggtacacca SEQUENCE ID NO. 8 50941 tcataacttcaaatctaatc gcggttggag gagcctctct aatcttggct ccggcaatta 51001 ctgggtatgttctacattgg aatattgctc tctcgacaat ctggggagta tcagcttatg 51061 gtattttcgtttttgggttt ttccttgcac aagttttatt ttcagaactg aacaggaaac 51121 gtcttcgcaagtggatttct ctcagaccta agggttggaa tgatgttcgt ttggctgtga 51181 tcattgctggatatcgcgag gatccttata tgttccagaa gtgcctcgag tctgtacgtg 51241 actctgattatggcaacgtt gcccgtctga tttgtgtgat tgacggtgat gaggacgatg 51301 atatgaggatggctgccgtt tacaaggcga tctacaatga taatatcaag aagcccgagt 51361 ttgttctgtgtgagtcagac gacaaggaag gtgaacgcat cgactctgat ttctctcgcg 51421 acatttgtgtcctccagcct catcgtggaa aacgggagtg tctttatact gggtttcaac 51481 ttgcaaagatggaccccagt gtcaatgctg tcgttctgat tgacagcgat accgttctcg 51541 agaaggatgctattctggaa gttgtatacc cacttgcatg cgatcccgag atccaagccg 51601 ttgcaggtgagtgtaagatt tggaacacag acactctttt gagtcttctc gtcgcttggc 51661 ggtactattctgcgttttgt gtggagagga gtgcccagtc ttttttcagg actgttcagt 51721 gcgttggggggccactgggt gcctacaaga ttgatatcat taaggagatt aaggacccct 51781 ggatttcccagcgctttctt ggtcagaagt gtacttacgg tgacgaccgc cggctaacca 51841 acgagatcttgatgcgtggt aaaaaggttg tgttcactcc atttgctgtt ggttggtctg 51901 acagtccgaccaatgtgttt cggtacatcg ttcagcagac ccgctggagt aagtcgtggt 51961 gccgcgaaatttggtacacc ctcttcgccg cgtggaagca cggtttgtct ggaatttggc 52021 tggcctttgaatgtttgtat caaattacat acttcttcct cgtgatttac ctcttttctc 52081 gcctagccgttgaggccgac cctcgcgccc agacagccac ggtgattgtg agcaccacgg 52141 ttgcattgattaagtgtggg tatttttcat tccgagccaa ggatattcgg gcgttttact 52201 ttgtgctttatacatttgtt tactttttct gtatgattcc ggccaggatt actgcaatga 52261 tgacgctttgggacattggc tgggatactc gcggtggaaa cgagaagcct tccgttggca 52321 cccgggtcgctctgtgggca aagcaatatc tcattgcata tatgtggtgg gccgcggttg 52381 ttggcgctggagtttacagc atcgtccata actggatgtt cgattggaat tctctttctt 52441 atcgttttgctttggttggt atttgttctt acattgtttt tattgttatt gtgctggtgg 52501 tttatttcaccggcaaaatt acgacttgga atttcacgaa gcttcagaag gagctaatcg 52561 aggatcgcgttctgtacgat gcaactacca atgctcagtc tgtgTGAttt ttcctgcaag

[0216]                                         +1® SEQUENCE ID NO. 9 1                  M  N  T  L  S  Q  A  I  K  A  Y  N  S  N  D  Y  Q  −18ATTTTTTAAGGACAGAAAATGAATACATTATCACAAGCAATAAAAGCATATAACAGCAATGACTATCAA 18 L  A  L  K  L  F  E  K  S  A  E  I  Y  G  R  K  I  V  E  F  Q  I  T  52TTAGCACTCAAATTATTTGAAAAGTCGGCGGAAATCTATGGACGGAAAATTGTTGAATTTCAAATTACC 41 K  C  Q  E  K  L  S  A  H  P  S  V  N  S  A  H  L  S  V  N  K  E  E 121AAATGCCAAGAAAAACTCTCAGCACATCCTTCTGTTAATTCAGCACATCTTTCTGTAAATAAAGAAGAA 64 K  V  N  V  C  D  S  P  L  D  I  A  T  Q  L  L  L  S  N  V  K  K  L 190AAAGTCAATGTTTGCGATAGTCCGTTAGATATTGCAACACAACTGTTACTTTCCAACGTAAAAAAATTA 87 V  L  S  D  S  E  K  N  T  L  K  N  K  W  K  L  L  T  E  K  K  S  E 259GTACTTTCTGACTCGGAAAAAAACACGTTAAAAAATAAATGGAAATTGCTCACTGAGAAGAAATCTGAA110 N  A  E  V  R  A  V  A  L  V  P  K  D  F  P  K  D  L  V  L  A  P  L 328AATGCGGAGGTAAGAGCGGTCGCCCTTGTACCAAAAGATTTTCCCAAAGATCTGGTTTTAGCGCCTTTA133 P  D  H  V  N  D  F  T  W  Y  K  K  R  K  K  R  L  G  I  K  P  E  H 397CCTGATCATGTTAATGATTTTACATGGTACAAAAAGCGAAAGAAAAGACTTGGCATAAAACCTGAACAT156 Q  H  V  G  L  S  I  I  V  T  T  F  N  R  P  A  I  L  S  I  T  L  A 466CAACATGTTGGTCTTTCTATTATCGTTACAACATTCAATCGACCAGCAATTTTATCGATTACATTAGCC179 C  L  V  N  Q  K  T  H  Y  P  F  E  V  I  V  T  D  D  G  S  Q  E  D 535TGTTTAGTAAACCAAAAAACACATTACCCGTTTGAAGTTATCGTGACAGATGATGGTAGTCAGGAAGAT202 L  S  P  I  I  R  Q  Y  E  N  K  L  D  I  R  Y  V  R  Q  K  D  N  G 604CTATCACCGATCATTCGCCAATATGAAAATAAATTGGATATTCGCTACGTCAGACAAAAAGATAACGGT225 F  Q  A  S  A  A  R  N  M  G  L  R  L  A  K  Y  D  F  I  G  L  L  D 673TTTCAAGCCAGTGCCGCTCGGAATATGGGATTACGCTTAGCAAAATATGACTTTATTGGCTTACTCGAC248 C  D  M  A  P  N  P  L  W  V  H  S  Y  V  A  E  L  L  E  D  D  D  L 742TGTGATATGGCGCCAAATCCATTATGGGTTCATTCTTATGTTGCAGAGCTATTAGAAGATGATGATTTA271 T  I  I  C  P  R  K  Y  I  D  T  Q  H  I  D  P  K  D  F  L  N  N  A 811ACAATCATTGGTCCAAGAAAATACATCGATACACAACATATTGACCCAAAAGACTTCTTAAATAACGCG294 S  L  L  E  S  L  P  E  V  K  T  N  N  S  V  A  A  K  G  E  G  T  V 880AGTTTGCTTGAATCATTACCAGAAGTGAAAACCAATAATAGTGTTGCCGCAAAAGGGGAAGGAACAGTT317 S  L  D  W  R  L  E  Q  F  E  K  T  E  N  L  R  L  S  D  S  P  F  R 949TCTCTGGATTGGCGCTTAGAACAATTCGAAAAAACAGAAAATCTCCGCTTATCCGATTCGCCTTTCCGT340 F  F  A  A  G  N  V  A  F  A  K  K  W  L  N  K  S  G  F  F  D  E  E 1018TTTTTTGCGGCGGGTAATGTTGCTTTCGCTAAAAAATGGCTAAATAAATCCGGTTTCTTTGATGAGGAA363 F  N  H  W  G  G  E  D  V  E  F  G  Y  R  L  F  R  Y  G  S  F  F  K 1087TTTAATCACTGGGGTGGAGAAGATGTGGAATTTGGATATCGCTTATTCCGTTACGGTAGTTTCTTTAAA386 T  I  D  G  I  M  A  Y  H  Q  E  P  P  G  K  E  N  E  T  D  R  E  A 1156ACTATTGATGGCATTATGGCCTACCATCAAGAGCCACCAGGTAAAGAAAATGAAACCGATCGTGAAGCG409 G  K  N  I  T  L  D  I  M  R  E  K  V  P  Y  I  Y  R  K  L  L  P  I 1225GGAAAAAATATTACGCTCGATATTATGAGAGAAAAGGTCCCTTATATCTATAGAAAACTTTTACCAATA432 E  D  S  H  I  N  R  V  P  L  V  S  I  Y  I  P  A  Y  N  C  A  N  Y 1294GAAGATTCGCATATCAATAGAGTACCTTTAGTTTCAATTTATATCCCAGCTTATAACTGTGCAAACTAT455 I  Q  R  C  V  D  S  A  L  N  Q  T  V  V  D  L  E  V  C  I  C  N  D 1363ATTCAACGTTGCGTAGATAGTGCACTGAATCAGACTGTTGTTGATCTCGAGGTTTGTATTTGTAACGAT478 G  S  T  D  N  T  L  E  V  I  N  K  L  Y  G  N  N  P  R  V  R  I  M 1432GGTTCAACAGATAATACCTTAGAAGTGATCAATAAGCTTTATGGTAATAATCCTAGGGTACGCATCATG501 S  K  P  N  G  G  I  A  S  A  S  N  A  A  V  S  F  A  K  G  Y  Y  I 1501TCTAAACCAAATGGCGGAATAGCCTCAGCATCAAATGCAGCCGTTTCTTTTGCTAAAGGTTATTACATT524 G  Q  L  D  S  D  D  Y  L  E  P  D  A  V  E  L  C  L  K  E  F  L  K 1570GGGCAGTTAGATTCAGATGATTATCTTGAGCCTGATGCAGTTGAACTGTGTTTAAAAGAATTTTTAAAA547 D  K  T  L  A  C  V  Y  T  T  N  R  N  V  N  P  D  G  S  L  I  A  N 1639GATAAAACGCTAGCTTGTGTTTATACCACTAATAGAAACGTCAATCCGGATGGTAGCTTAATCGCTAAT570 G  Y  N  W  P  E  F  S  R  E  K  L  T  T  A  M  I  A  H  H  F  R  M 1708GGTTACAATTGGCCAGAATTTTCACGAGAAAAACTCACAACGGCTATGATTGCTCACCACTTTAGAATC593 F  T  I  R  A  W  H  L  T  D  G  F  N  E  K  I  E  N  A  V  D  Y  D 1777TTCACGATTAGAGCTTGGCATTTAACTGATGGATTCAATGAAAAAATTGAAAATGCCGTAGACTATGAC616 M  F  L  K  L  S  E  V  G  K  F  K  H  L  N  K  I  C  Y  N  R  V  L 1846ATGTTCCTCAAACTCAGTGAAGTTGGAAAATTTAAACATCTTAATAAAATCTGCTATAACCGTGTATTA639 H  G  D  N  T  S  I  K  K  L  G  I  Q  K  K  N  H  F  V  V  V  N  Q 1915CATGGTGATAACACATCAATTAAGAAACTTGGCATTCAAAAGAAAAACCATTTTGTTGTAGTCAATCAG662 S  L  N  R  Q  G  I  T  Y  Y  N  Y  D  E  F  D  D  L  D  E  S  R  K 1984TCATTAAATAGACAAGGCATAACTTATTATAATTATGACGAATTTGATGATTTAGATGAAAGTAGAAAG685 Y  I  F  N  K  T  A  E  Y  Q  E  E  I  D  I  L  K  D  I  K  I  I  Q 2053TATATTTTCAATAAAACCGCTGAATATCAAGAAGAGATTGATATCTTAAAAGATATTAAAATCATCCAC708 N  K  D  A  K  I  A  V  S  I  F  Y  P  N  T  L  N  G  L  V  K  K  L 2122AATAAAGATGCCAAAATCGCAGTCAGTATTTTTTATCCCAATACATTAAACGGCTTAGTGAAAAAACTA731 N  N  I  I  E  Y  N  K  N  I  F  V  I  V  L  H  V  D  K  N  H  L  T 2191AACAATATTATTGAATATAATAAAAATATATTCGTTATTGTTCTACATGTTGATAAGAATCATCTTACA754 P  D  I  K  K  E  I  L  A  F  Y  H  K  H  Q  V  N  I  L  L  N  N  C 2260CCAGATATCAAAAAAGAAATACTAGCCTTCTATCATAAACATCAAGTGAATATTTTACTAAATAATGAT777 I  S  Y  Y  T  S  N  R  L  I  K  T  E  A  H  L  S  N  I  N  K  L  S 2329ATCTCATATTACACGAGTAATAGATTAATAAAAACTGAGGCGCATTTAAGTAATATTAATAAATTAAGT800 Q  L  N  L  N  C  E  Y  I  I  F  D  N  H  D  S  L  F  V  K  N  D  S 2398CAGTTAAATCTAAATTGTGAATACATCATTTTTGATAATCATGACAGCCTATTCGTTAAAAATGACAGC823 Y  A  Y  M  K  K  Y  D  V  G  M  N  F  S  A  L  T  H  D  W  I  E  K 2467TATGCTTATATGAAAAAATATGATGTCGGCATGAATTTCTCAGCATTAACACATGATTGGATCGAGAAA846 I  N  A  H  P  P  F  K  K  L  I  K  T  Y  F  N  D  N  D  L  K  S  M 2536ATCAATGCGCATCCACCATTTAAAAAGCTCATTAAAACTTATTTTAATGACAATGACTTAAAAAGTATG869 N  V  K  G  A  S  Q  G  M  F  M  T  Y  A  L  A  H  E  L  L  T  I  I 2605AATGTGAAAGGGGCATCACAAGGTATGTTTATGACGTATGCGCTAGCGCATGAGCTTCTGACGATTATT892 K  E  V  I  T  S  C  Q  S  I  D  S  V  P  E  Y  N  T  E  D  I  W  F 2674AAAGAAGTCATCACATCTTGCCAGTCAATTGATAGTGTGCCAGAATATAACACTGAGGATATTTGGTTC915 Q  F  A  L  L  I  L  E  K  K  T  G  H  V  F  N  K  T  S  T  L  T  Y 2743CAATTTGCACTTTTAATCTTAGAAAAGAAAACCGGCCATGTATTTAATAAAACATCGACCCTGACTTAT938 M  P  W  E  R  K  L  Q  W  T  N  E  Q  I  E  S  A  K  R  G  E  N  I 2812ATGCCTTGGGAACGAAAATTACAATGGACAAATGAACAAATTGAAAGTGCAAAAAGAGGAGAAAATATA961  P  V  N  K  F  I  I  N  S  I  T  L  * 2881CCTGTTAACAAGTTCATTATTAATAGTATAACTCTATAA

What we claim is:
 1. A purified nucleic acid segment comprising a codingregion encoding enzymatically active hyaluronate synthase.
 2. Thepurified nucleic acid segment of claim 1, wherein the purified nucleicacid segment encodes the Streptococcus equisimilis hyaluronate synthaseof SEQ ID NO:2.
 3. The purified nucleic acid segment of claim 1, whereinthe purified nucleic acid segment comprises a nucleotide sequence inaccordance with SEQ ID NO:1.
 4. A purified nucleic acid segment having acoding region encoding enzymatically active hyaluronate synthase,wherein the purified nucleic acid segment is capable of hybridizing tothe nucleotide sequence of SEQ ID NO:1.
 5. A purified nucleic acidsegment having a coding region encoding enzymatically active hyaluronatesynthase, wherein the purified nucleic acid segment has semiconservativeor conservative amino codon acid changes when compared to the nucleotidesequence of SEQ ID NO:1.
 6. A recombinant vector selected from the groupconsisting of a plasmid, cosmid, phage, or virus vector and wherein therecombinant vector further comprises a purified nucleic acid segmenthaving a coding region encoding enzymatically active hyaluronansynthase.
 7. The recombinant vector of claim 5, wherein the purifiednucleic acid segment encodes the Streptococcus equisimilis hyaluronansynthase of SEQ ID NO:2.
 8. The recombinant vector of claim 6, whereinthe purified nucleic acid segment comprises a nucleotide sequence inaccordance with SEQ ID NO:1.
 9. The recombinant vector of claim 6,wherein the plasmid further comprises an expression vector.
 10. Therecombinant vector of claim 9, wherein the expression vector comprises apromoter operatively linked to the enzymatically active Streptococcusequisimilis hyaluronan synthase coding region.
 11. A recombinant hostcell, wherein the recombinant host cell is a prokaryotic celltransformed with a recombinant vector comprising a purified nucleic acidsegment having a coding region encoding enzymatically active hyaluronansynthase.
 12. The recombinant host cell of claim 11, wherein thepurified nucleic acid segment encodes the Streptococcus equisimilishyaluronan synthase of SEQ ID NO:2.
 13. The recombinant host cell ofclaim 11, wherein the purified nucleic acid segment comprises anucleotide sequence in accordance with SEQ ID NO:1.
 14. The recombinanthost cell of claim 13, wherein the host cell produces hyaluronic acid.15. The recombinant host cell of claim 11, wherein the enzymaticallyactive hyaluronan synthase is capable of producing a hyaluronic acidpolymer having a modified structure.
 16. The recombinant host cell ofclaim 11, wherein the enzymatically active hyaluronan synthase iscapable of producing a hyaluronic acid polymer having a modified sizedistribution.
 17. A recombinant host cell, wherein the recombinant hostcell is a eukaryotic cell transfected with a recombinant vectorcomprising a purified nucleic acid segment having a coding regionencoding enzymatically active hyaluronan synthase.
 18. The recombinanthost cell of claim 17, wherein the purified nucleic acid segment encodesthe Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:2. 19.The recombinant host cell of claim 17, wherein the purified nucleic acidsegment comprises a nucleotide sequence in accordance with SEQ ID NO:1.20. The recombinant host cell of claim 19, wherein the host cellproduces hyaluronic acid.
 21. The recombinant host cell of claim 17,wherein the enzymatically active hyaluronan synthase is capable ofproducing a hyaluronic acid polymer having a modified structure.
 22. Therecombinant host cell of claim 17, wherein the enzymatically activehyaluronan synthase is capable of producing a hyaluronic acid polymerhaving a modified size distribution.
 23. A recombinant host cell,wherein the recombinant host cell is electroporated to introduce arecombinant vector into the recombinant host cell, wherein therecombinant vector comprises a purified nucleic acid segment having acoding region encoding enzymatically active hyaluronan synthase.
 24. Therecombinant host cell of claim 23, wherein the purified nucleic acidsegment encodes the Streptococcus equisimilis hyaluronan synthase of SEQID NO:2.
 25. The recombinant host cell of claim 23, wherein the purifiednucleic acid segment comprises a nucleotide sequence in accordance withSEQ ID NO:1.
 26. The recombinant host cell of claim 25, wherein the hostcell produces hyaluronic acid.
 27. The recombinant host cell of claim23, wherein the enzymatically active hyaluronan synthase is capable ofproducing a hyaluronic acid polymer having a modified structure.
 28. Therecombinant host cell of claim 23, wherein the enzymatically activeStreptococcus equisimilis hyaluronan synthase is capable of producing ahyaluronic acid polymer having a modified size distribution.
 29. Arecombinant host cell, wherein the recombinant host cell is transducedwith a recombinant vector comprising a purified nucleic acid segmenthaving a coding region encoding enzymatically active Streptococcusequisimilis hyaluronan synthase.
 30. The recombinant host cell of claim29, wherein the purified nucleic acid segment encodes the Streptococcusequisimilis hyaluronan synthase of SEQ ID NO:2.
 31. The recombinant hostcell of claim 29, wherein the purified nucleic acid segment comprises anucleotide sequence in accordance with SEQ ID NO:1.
 32. The recombinanthost cell of claim 31, wherein the host cell produces hyaluronic acid.33. The recombinant host cell of claim 29, wherein the enzymaticallyactive hyaluronan synthase is capable of producing a hyaluronic acidpolymer having a modified structure.
 34. The recombinant host cell ofclaim 29, wherein the enzymatically active hyaluronan synthase iscapable of producing a hyaluronic acid polymer having a modified sizedistribution.
 35. A purified composition, wherein the purifiedcomposition comprises an enzymatically active hyaluronan synthasepolypeptide.
 36. A purified composition, wherein the purifiedcomposition comprises a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2.
 37. A method for detecting a DNA species,comprising the steps of: obtaining a DNA sample; contacting the DNAsample with a purified nucleic acid segment in accordance with SEQ IDNO:1; hybridizing the DNA sample and the purified nucleic acid segmentthereby forming a hybridized complex; and detecting the complex.
 38. Amethod for detecting a bacterial cell that expresses mRNA encodingStreptococcus equisimilis hyaluronan synthase, comprising the steps of:obtaining a bacterial cell sample; contacting at least one nucleic acidfrom the bacterial cell sample with purified nucleic acid segment inaccordance with SEQ ID NO:1; hybridizing the at least one nucleic acidand the purified nucleic acid segment thereby forming a hybridizedcomplex; and detecting the hybridized complex, wherein the presence ofthe hybridized complex is indicative of a bacterial strain thatexpresses mRNA encoding Streptococcus equisimilis hyaluronan synthase.39. A method for producing hyaluronic acid, comprising the steps of:introducing a purified nucleic acid segment having a coding regionencoding enzymatically active hyaluronan synthase into a host organism,wherein the host organism contains nucleic acid segments encodingenzymes which produce UDP-GlcNAc and UDP-GlcA; growing the host organismin a medium to secrete hyaluronic acid; and recovering the secretedhyaluronic acid.
 40. The method according to claim 39, wherein the stepof recovering the hyaluronic acid comprises extracting the secretedhyaluronic acid from the medium.
 41. The method according to claim 40,further comprising the step of purifying the extracted hyaluronic acid.42. The method according to claim 39, wherein in the step of growing thehost organism, the host organism secretes a structurally modifiedhyaluronic acid.
 43. The method according to claim 39, wherein in thestep of growing the host organism, the host organism secretes ahyaluronic acid having a modified size.
 44. A pharmaceutical compositioncomprising a preselected pharmaceutical drug and an effective amount ofhyaluronic acid produced by hyaluronan synthase.
 45. The pharmaceuticalcomposition of claim 44, wherein the hyaluronic acid is produced by theStreptococcus equisimilis hyaluronan synthase of SEQ ID NO:2.
 46. Thepharmaceutical composition according to claim 44, wherein the molecularweight of the hyaluronic acid is modified thereby producing a modifiedmolecular weight pharmaceutical composition capable of evading an immuneresponse.
 47. The pharmaceutical composition according to claim 44,wherein the molecular weight of the hyaluronic acid is modified therebyproducing a modified molecular weight pharmaceutical composition capableof targeting a specific tissue or cell type within the patient having anaffinity for the modified molecular weight pharmaceutical composition.48. A purified and isolated nucleic acid sequence encoding enzymaticallyactive hyaluronan synthase, the nucleic acid sequence selected from thegroup consisting of: (a) the nucleic acid sequence in accordance withSEQ ID NO:1; (b) complementary nucleic acid sequences to the nucleicacid sequence in accordance with SEQ ID NO:1; (c) nucleic acid sequenceswhich will hybridize to the nucleic acid in accordance with SEQ ID NO:1;(d) nucleic acid sequences which will hybridize to the complementarynucleic acid sequences of SEQ ID NO:1; and (e) nucleic acid sequenceswhich will hybridize to PCR probes selected from the group consisting ofPCR probes of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
 49. Apurified and isolated nucleic acid segment consisting essentially of anucleic acid segment encoding enzymatically active hyaluronan synthase.50. A procaryotic or eucaryotic host cell transformed or transfectedwith an isolated nucleic acid segment according to claim 1, 2, or 3 in amanner allowing the host cell to express hyaluronic acid.
 51. Anisolated nucleic acid segment consisting essentially of a nucleic acidsegment encoding hyaluronan synthase having a nucleic acid segmentsufficiently duplicative of the nucleic acid segment in accordance ofSEQ ID NO:1 to allow possession of the biological property of encodingfor Streptococcus equisimilis hyaluronan synthase.
 52. A cDNA sequenceaccording to claim
 51. 53. A procaryotic or eucaryotic host celltransformed or transfected with a nucleic acid segment according toclaim 51 in a manner allowing the host cell to express hyaluronic acid.54. A purified nucleic acid segment having a coding region encodingenzymatically active hyaluronan synthase, wherein the purified nucleicacid segment is capable of hybridizing to the nucleotide sequence inaccordance with SEQ ID NO:1.
 55. A purified nucleic acid segmentaccording to SEQ ID NO:3 capable of hybridizing to SEQ ID NO:1.
 56. Apurified nucleic acid segment according to SEQ ID NO:4 capable ofhybridizing to SEQ ID NO:1.
 57. A purified nucleic acid segmentaccording to SEQ ID NO:5 capable of hybridizing to SEQ ID NO:1.
 58. Apurified nucleic acid segment according to SEQ ID NO:4 capable ofhybridizing to SEQ ID NO:1.
 59. A purified nucleic acid segment having acoding region encoding enzymatically active hyaluronate synthase, thepurified nucleic acid segment selected from the group consisting of: (A)the nucleic acid segment according to SEQ ID NO:2; (B) the nucleotidesequence in accordance with SEQ ID NO:1; (C) nucleic acid segments whichhybridize to the nucleic acid segments defined in (A) or (B) orfragments thereof; and (D) nucleic acid segments which but for thedegeneracy of the genetic code, or encoding of functionally equivalentamino acids, would hybridize to the nucleic acid segments defined in(A), (B), and (C).